( F ) Immunofluorescence analysis of the old APPPS1 slice in co - culture ( 14 DIV ) treated with cytochalasin D ( CytoD ) and vehicle control ( Ctr ) and immunostained with M3 . 2 ( green ) and CD68 ( red ) . Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance . Scale bar : 50 µm . ( G ) Quantitative analysis of core - only plaques in the old APPPS1 slice in co - culture ( 14 DIV ) treated with CytoD and Ctr reveals a decreased number of core - only plaques upon CytoD treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques . The values represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * * P < 0 . 001 ) .

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

(F) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) treated with cytochalasin D (CytoD) and vehicle control (Ctr) and immunostained with M3.2 (green) and CD68 (red). Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance. Scale bar: 50 µm.(G) Quantitative analysis of core-only plaques in the old APPPS1 slice in co-culture (14 DIV) treated with CytoD and Ctr reveals a decreased number of core-only plaques upon CytoD treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).


Either young WT ( A , B ) or old APPPS1 ( C - E ) tissue was treated with clodronate ( Clo ) to remove CD68 positive cells or vehicle Ctr from 1 until 7 DIV . Treatment was stopped and subsequently old ( A , B ) or young ( C - E ) tissue was added to the culture as schematically indicated in A and C . and analyzed 14 days after . Removal of CD68 positive cells in either young WT or old APPPS1 tissue prevents amyloid plaque clearance in the co - culture model . ( A ) Immunofluorescence analysis of the old APPPS1 slice co - cultured with the young WT slice pre - treated with Clo and Ctr and immunostained with CD68 ( red ) and M3 . 2 ( green ) . Scale bar : 50 µm . ( B ) Quantitative analysis of core - only plaques in the old APPPS1 tissue co - cultured with the young WT tissue pre - treated with Clo and Ctr as indicated in A reveals a decreased number of core - only plaques upon Clo treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * * P < 0 . 001 ) . ( C ) Immunofluorescence analysis of the old APPPS1 slice treated with Clo and Ctr and subsequently co - cultured with the young WT slice and immunostained with CD68 ( red ) and M3 . 2 ( green ) . Scale bar : 50 µm . ( D ) Area of CD68 positive cells ( CD68 coverage ) in the old APPPS1 tissue treated with Clo and Ctr and subsequently co - cultured with the young WT tissue as indicated in C . CD68 coverage is reduced upon Clo treatment . The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * * P < 0 . 001 ) . ( E ) Quantitative analysis of core - only plaques in the old APPPS1 tissue treated with Clo and Ctr and subsequently co - cultured with the young WT tissue as indicated in C reveals a decreased number of core - only plaques upon Clo treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * P < 0 . 01 ) .

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

Either young WT (A, B) or old APPPS1 (C-E) tissue was treated with clodronate (Clo) to remove CD68 positive cells or vehicle Ctr from 1 until 7 DIV. Treatment was stopped and subsequently old (A, B) or young (C-E) tissue was added to the culture as schematically indicated in A and C. and analyzed 14 days after. Removal of CD68 positive cells in either young WT or old APPPS1 tissue prevents amyloid plaque clearance in the co-culture model.(A) Immunofluorescence analysis of the old APPPS1 slice co-cultured with the young WT slice pre-treated with Clo and Ctr and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.(B) Quantitative analysis of core-only plaques in the old APPPS1 tissue co-cultured with the young WT tissue pre-treated with Clo and Ctr as indicated in A reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).(C) Immunofluorescence analysis of the old APPPS1 slice treated with Clo and Ctr and subsequently co-cultured with the young WT slice and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.(D) Area of CD68 positive cells (CD68 coverage) in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C. CD68 coverage is reduced upon Clo treatment. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).(E) Quantitative analysis of core-only plaques in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (**P < 0.01).


( A ) Immunofluorescence analysis of the old APPPS1 slice in co - culture with the young CX3CR1 + / GFP slice ( 14 DIV ) and immunostained using GFP ( green ) , CD68 ( red ) and M3 . 2 ( blue ) reveals no co - localization of young , GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques . A GFP antibody was used to amplify the signal of GFP - expressing young microglial cells . ( B ) Immunofluorescence analysis of the old APPPS1 / CX3CR1 + / GFP slice in co - culture with the young WT slice ( 14 DIV ) and immunostained using GFP ( green ) , CD68 ( red ) and M3 . 2 ( blue ) reveals co - localization of old , GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques . A GFP antibody was used to amplify the signal of GFP - expressing old microglial cells . Scale bar : 10 µm ( A and B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of the old APPPS1 slice in co-culture with the young CX3CR1+/GFP slice (14 DIV) and immunostained using GFP (green), CD68 (red) and M3.2 (blue) reveals no co-localization of young, GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques. A GFP antibody was used to amplify the signal of GFP-expressing young microglial cells.(B) Immunofluorescence analysis of the old APPPS1/CX3CR1+/GFP slice in co-culture with the young WT slice (14 DIV) and immunostained using GFP (green), CD68 (red) and M3.2 (blue) reveals co-localization of old, GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques. A GFP antibody was used to amplify the signal of GFP-expressing old microglial cells. Scale bar: 10 µm (A and B).


( C ) 3D reconstruction of the old APPPS1 / CX3CR1 + / GFP slice in co - culture with the young WT slice ( 10 DIV ) and immunostained using GFP ( green ) and M3 . 2 ( red ) . GFP positive old microglial cells surrounding amyloid plaques exhibit intracellular M3 . 2 positive immunoreactivity indicative of Aβ uptake . Overview at low magnification is shown in the left panel . Scale bar : 15 µm . A detailed plaque is shown at higher magnification in the right panels . Scale bar : 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 O O O O O O O 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) 3D reconstruction of the old APPPS1/CX3CR1+/GFP slice in co-culture with the young WT slice (10 DIV) and immunostained using GFP (green) and M3.2 (red). GFP positive old microglial cells surrounding amyloid plaques exhibit intracellular M3.2 positive immunoreactivity indicative of Aβ uptake. Overview at low magnification is shown in the left panel. Scale bar: 15 µm. A detailed plaque is shown at higher magnification in the right panels. Scale bar: 10 µm.


( D ) Immunofluorescence analysis of young CX3CR1 + / GFP and old APPPS1 / CX3CR1 + / GFP slices freshly cut ( 0 DIV ) , cultured alone or in co - culture ( 10 DIV ) and immunostained using GFP ( green ) shows alterations in microglial morphology of old cells upon culturing . Upper - left panels are enlargements of boxed regions . A GFP antibody was used to amplify the signal of GFP - expressing old microglial cells . Scale bars : 50 µm . ( E ) Quantitative analysis of amoeboid microglial cells in young CX3CR1 + / GFP ( white bars ) and old APPPS1 / CX3CR1 + / GFP ( green bars ) brain slices freshly cut ( 0 DIV ) , cultured alone or in co - culture ( 10 DIV ) . The values are expressed as percentages of amoeboid microglia from the total number of microglial cells . The values represent mean ± SEM from 3 independent experiments , each experiment including at least 250 cells per condition ( * P < 0 . 05 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of young CX3CR1+/GFP and old APPPS1/CX3CR1+/GFP slices freshly cut (0 DIV), cultured alone or in co-culture (10 DIV) and immunostained using GFP (green) shows alterations in microglial morphology of old cells upon culturing. Upper-left panels are enlargements of boxed regions. A GFP antibody was used to amplify the signal of GFP-expressing old microglial cells. Scale bars: 50 µm.(E) Quantitative analysis of amoeboid microglial cells in young CX3CR1+/GFP (white bars) and old APPPS1/CX3CR1+/GFP (green bars) brain slices freshly cut (0 DIV), cultured alone or in co-culture (10 DIV). The values are expressed as percentages of amoeboid microglia from the total number of microglial cells. The values represent mean ± SEM from 3 independent experiments, each experiment including at least 250 cells per condition (*P < 0.05, ***P < 0.001).


( A ) Old APPPS1 tissue was cultured alone ( old alone , white bars ) or incubated with conditioned media ( CM ) collected from young WT slices ( old alone + young CM , grey bars ) . Analysis included co - culture of old APPPS1 and young WT brain slices ( old co - culture , black bars ) as a positive control and old APPPS1 tissue incubated with conditioned media collected from old APPPS1 slices ( old alone + old CM , white squared bars ) as a negative control . Quantitative analysis at 7 , 11 and 14 DIV reveals increased numbers of core - only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices , fully recapitulating core - only plaque numbers observed in the co - culture . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including at least 2 independent slice culture dishes ( n . s . = not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Old APPPS1 tissue was cultured alone (old alone, white bars) or incubated with conditioned media (CM) collected from young WT slices (old alone + young CM, grey bars). Analysis included co-culture of old APPPS1 and young WT brain slices (old co-culture, black bars) as a positive control and old APPPS1 tissue incubated with conditioned media collected from old APPPS1 slices (old alone + old CM, white squared bars) as a negative control. Quantitative analysis at 7, 11 and 14 DIV reveals increased numbers of core-only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices, fully recapitulating core-only plaque numbers observed in the co-culture. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including at least 2 independent slice culture dishes (n.s.= not significant, **P <0.01, ***P < 0.001).


( B ) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices previously treated with clodronate ( old alone + young CM + Clo , grey bars ) and vehicle control ( old alone + young CM + Ctr , white bars ) . Quantitative analysis at 7 , 11 and 14 DIV reveals decreased numbers of core - only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices pre - treated with Clo . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including at least 2 independent slice culture dishes ( n . s . = not significant , * * * P < 0 . 001 ) .

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 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 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

(B) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices previously treated with clodronate (old alone + young CM + Clo, grey bars ) and vehicle control (old alone + young CM + Ctr, white bars). Quantitative analysis at 7, 11 and 14 DIV reveals decreased numbers of core-only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices pre-treated with Clo. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including at least 2 independent slice culture dishes (n.s.= not significant, ***P < 0.001).


( C ) Old APPPS1 tissue was incubated with conditioned media collected from cultured WT primary microglial cells ( old alone + MG CM , grey bar ) or with unconditioned slice culture media as a control ( old alone + Ctr , white bar ) . Quantitative analysis of core - only plaques at 14 DIV reveals enhanced amyloid plaque clearance upon incubation of the old APPPS1 tissue with conditioned media collected from cultured primary microglia . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * P < 0 . 05 ) .

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

(C) Old APPPS1 tissue was incubated with conditioned media collected from cultured WT primary microglial cells (old alone + MG CM, grey bar) or with unconditioned slice culture media as a control (old alone + Ctr, white bar). Quantitative analysis of core-only plaques at 14 DIV reveals enhanced amyloid plaque clearance upon incubation of the old APPPS1 tissue with conditioned media collected from cultured primary microglia. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).


( A - F ) Immunofluorescence analysis of the old APPPS1 slice ( 14 DIV ) treated with vehicle Ctr in A , anti - inflammatory factors IL - 10 and TGF - β in B and C and pro - inflammatory factors IL - 6 , IL - 12 / p40 and GM - CSF in D , E and F , respectively and immunostained with CD68 ( red ) and M3 . 2 ( green ) . In contrast to increased number of core - only plaques upon GM - CSF treatment , other factors tested did not induce amyloid plaque clearance , resulting in a better preservation of the M3 . 2 positive halos of Aβ surrounding dense core of amyloid plaques . Scale bar : 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Immunofluorescence analysis of the old APPPS1 slice (14 DIV) treated with vehicle Ctr in A, anti-inflammatory factors IL-10 and TGF-β in B and C and pro-inflammatory factors IL-6, IL-12/p40 and GM-CSF in D, E and F, respectively and immunostained with CD68 (red) and M3.2 (green). In contrast to increased number of core-only plaques upon GM-CSF treatment, other factors tested did not induce amyloid plaque clearance, resulting in a better preservation of the M3.2 positive halos of Aβ surrounding dense core of amyloid plaques. Scale bar: 50 µm.


( A ) CD68 coverage in the old APPPS1 tissue ( 14 DIV ) treated with GM - CSF and vehicle Ctr . CD68 coverage is increased upon treatment with GM - CSF . The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * P < 0 . 05 ) .

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

(A) CD68 coverage in the old APPPS1 tissue (14 DIV) treated with GM-CSF and vehicle Ctr. CD68 coverage is increased upon treatment with GM-CSF. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).


( B ) Quantitative analysis of core - only plaques in the old APPPS1 tissue ( 14 DIV ) treated with GM - CSF and Ctr reveals an increased number of core - only plaques upon treatment with GM - CSF . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including 3 independent slice culture dishes ( * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) treated with GM-CSF and Ctr reveals an increased number of core-only plaques upon treatment with GM-CSF. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).


( C ) CD68 coverage in the old APPPS1 tissue ( 14 DIV ) incubated with conditioned media collected from young WT slices ( young CM ) or with non - conditioned slice culture media ( Ctr ) . CD68 coverage is increased upon incubation of the APPPS1 tissue with the young CM . The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD68 coverage in the old APPPS1 tissue (14 DIV) incubated with conditioned media collected from young WT slices (young CM) or with non-conditioned slice culture media (Ctr). CD68 coverage is increased upon incubation of the APPPS1 tissue with the young CM. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (**P < 0.01).


( D ) Immunofluorescence analysis of the old APPPS1 slice cultured alone ( old alone ) or in co - culture with young GM - CSF - / - brain slices ( old co - culture ) at 14 DIV and immunostained with CD68 ( red ) and M3 . 2 ( green ) reveals an increased number of core - only plaques upon co - culturing with young GM - CSF - / - brain slices . Scale bar : 50 µm . ( E ) Quantitative analysis of core - only plaques in the old APPPS1 slice cultured alone or in co - culture with young GM - CSF - / - brain slices ( 14 DIV ) reveals an increased number of core - only plaques upon co - culturing of old APPPS1 brain slices together with young GM - CSF - / - brain slices . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques . The values represent mean ± SEM from 3 independent experiments , each experiment including 3 independent slice culture dishes ( * * * P < 0 . 001 ) . ( F ) CD68 coverage in the old APPPS1 tissue ( 14 DIV ) cultured alone or in co - culture with young GM - CSF - / - brain slices . CD68 coverage is increased upon co - culturing of old APPPS1 brain slices together with young GM - CSF - / - brain slices . The values are normalized to CD68 coverage of the old slice cultured alone and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * P < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of the old APPPS1 slice cultured alone (old alone) or in co-culture with young GM-CSF-/- brain slices (old co-culture) at 14 DIV and immunostained with CD68 (red) and M3.2 (green) reveals an increased number of core-only plaques upon co-culturing with young GM-CSF-/- brain slices. Scale bar: 50 µm.(E) Quantitative analysis of core-only plaques in the old APPPS1 slice cultured alone or in co-culture with young GM-CSF-/- brain slices (14 DIV) reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).(F) CD68 coverage in the old APPPS1 tissue (14 DIV) cultured alone or in co-culture with young GM-CSF-/- brain slices. CD68 coverage is increased upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are normalized to CD68 coverage of the old slice cultured alone and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).


( A ) Immunofluorescence analysis of the old APPPS1 slice ( 14 DIV ) treated with GM - CSF and vehicle Ctr and immunostained with CD68 ( red ) , cell proliferation marker Ki67 ( green ) and M3 . 2 ( blue ) . Scale bar : 50 µm . ( B ) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue ( 14 DIV ) treated with GM - CSF and Ctr reveals increased microglial proliferation upon treatment with GM - CSF . The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 2 independent experiments , each experiment including 4 independent slice culture dishes ( * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of the old APPPS1 slice (14 DIV) treated with GM-CSF and vehicle Ctr and immunostained with CD68 (red), cell proliferation marker Ki67 (green) and M3.2 (blue). Scale bar: 50 µm.(B) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue (14 DIV) treated with GM-CSF and Ctr reveals increased microglial proliferation upon treatment with GM-CSF. The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 2 independent experiments, each experiment including 4 independent slice culture dishes (***P < 0.001).


( C ) Immunofluorescence analysis of the old APPPS1 tissue cultured alone or in co - culture with young WT brain slices ( 14 DIV ) and immunostained with CD68 ( red ) , Ki67 ( green ) and M3 . 2 ( blue ) Scale bar : 50 µm . ( D ) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue cultured alone or in co - culture with young brain slices ( 14 DIV ) reveals increased microglial proliferation upon co - culturing of old and young brain slices . The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of the old APPPS1 tissue cultured alone or in co-culture with young WT brain slices (14 DIV) and immunostained with CD68 (red), Ki67 (green) and M3.2 (blue) Scale bar: 50 µm.(D) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue cultured alone or in co-culture with young brain slices (14 DIV) reveals increased microglial proliferation upon co-culturing of old and young brain slices. The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (**P < 0.01).


( E ) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices and in addition treated with proliferation inhibitor AraC ( young CM + AraC ) or vehicle Ctr ( young CM + Ctr ) . Immunofluorescence analysis was performed at 14 DIV using CD68 ( red ) and M3 . 2 ( green ) . Scale bar : 50 µm . ( F ) Quantitative analysis of core - only plaques in the old APPPS1 tissue ( 14 DIV ) incubated with conditioned media from young WT slices and in addition treated with AraC or Ctr reveals a decreased number of core - only plaques upon AraC treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each including 2 independent slice culture dishes ( * * * P < 0 . 001 ) .

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

(E) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices and in addition treated with proliferation inhibitor AraC (young CM + AraC) or vehicle Ctr (young CM + Ctr). Immunofluorescence analysis was performed at 14 DIV using CD68 (red) and M3.2 (green). Scale bar: 50 µm.(F) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) incubated with conditioned media from young WT slices and in addition treated with AraC or Ctr reveals a decreased number of core-only plaques upon AraC treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each including 2 independent slice culture dishes (***P < 0.001).


A . Hcp release assay . HA - tagged Hcp ( HcpHA ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 wild - type ( WT ) , ΔtssM cells or ΔtssM cells carrying the AHT - inducible FLAG - tagged tssM - borne plasmid ( tssM + ) treated ( bulgecin ) or not ( NT ) with bulgecin A prior to tssM gene induction . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and the periplasmic TolB protein ( control for cell lysis ) , HcpHA and FLTssM were immunodetected using anti - TolB ( middle panel ) , anti - HA ( lower panel ) and anti - FLAG ( upper panel ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in duplicate and a representative result is shown .

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

A. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT), ΔtssM cells or ΔtssM cells carrying the AHT-inducible FLAG-tagged tssM-borne plasmid (tssM+) treated (bulgecin) or not (NT) with bulgecin A prior to tssM gene induction. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis), HcpHA and FLTssM were immunodetected using anti-TolB (middle panel), anti-HA (lower panel) and anti-FLAG (upper panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.


B . Hcp release assay . HA - tagged Hcp ( HcpHA ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 wild - type ( WT ) cells before washing cells ( before wash ) and after washing and growth ( after wash ) in absence ( NT ) or presence ( bulgecin ) of bulgecin A . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and the periplasmic TolB protein ( control for cell lysis ) and HcpHA were immunodetected using anti - TolB ( upper panel ) and anti - HA ( lower panel ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in duplicate and a representative result is shown .

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

B. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT) cells before washing cells (before wash) and after washing and growth (after wash) in absence (NT) or presence (bulgecin) of bulgecin A. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis) and HcpHA were immunodetected using anti-TolB (upper panel) and anti-HA (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.


Hcp release assay . FLAG - tagged Hcp ( HcpFL ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 cells of the indicated strains . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and TolB and HcpFL were immunodetected using anti - TolB ( upper panel ) and anti - FLAG ( lower panel ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

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

Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 cells of the indicated strains. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB and HcpFL were immunodetected using anti-TolB (upper panel) and anti-FLAG (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


A . Hcp release assay . FLAG - tagged Hcp ( HcpFL ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 WT , ΔmltE cells or ΔmltE cells producing wild - type ( mltE + ) or E64Q mutant ( mltEE64Q ) VSV - G - tagged MltE ( MltEV ) from arabinose - inducible plasmids . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and TolB , HcpFL and MltEV were immunodetected using anti - TolB ( upper panel ) , anti - FLAG ( middle panel ) and anti - VSV - G ( lower panel ) antibodies , respectively . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 WT, ΔmltE cells or ΔmltE cells producing wild-type (mltE+) or E64Q mutant (mltEE64Q) VSV-G-tagged MltE (MltEV) from arabinose-inducible plasmids. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB, HcpFL and MltEV were immunodetected using anti-TolB (upper panel), anti-FLAG (middle panel) and anti-VSV-G (lower panel) antibodies, respectively. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


B . Anti - bacterial activity . E . coli K - 12 prey cells ( W3110 gfp + , kanR ) were mixed with the indicated attacker cells , spotted onto Sci - 1 inducing medium ( SIM ) agar plates and incubated for 4 hours at 37 ° C . The image of a representative bacterial spot and the average and standard deviation ( n = 3 ) of the relative fluorescence of the bacterial mixture ( in arbitrary unit , AU ) are shown in the upper graph . The number of recovered E . coli prey cells ( counted on selective kanamycin medium ) is indicated in the lower graph ( in log10 of colony - forming unit ( cfu ) ) . The black , dark grey and light grey circles indicate values from three independent assays , and the average is indicated by the bar . The experiment was performed in triplicate and a representative result is shown . Asterisks indicate significant differences compared to the wild - type attacker strain ( NS , non significant ; * * * , p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Anti-bacterial activity. E. coli K-12 prey cells (W3110 gfp+, kanR) were mixed with the indicated attacker cells, spotted onto Sci-1 inducing medium (SIM) agar plates and incubated for 4 hours at 37°C. The image of a representative bacterial spot and the average and standard deviation (n=3) of the relative fluorescence of the bacterial mixture (in arbitrary unit, AU) are shown in the upper graph. The number of recovered E. coli prey cells (counted on selective kanamycin medium) is indicated in the lower graph (in log10 of colony-forming unit (cfu)). The black, dark grey and light grey circles indicate values from three independent assays, and the average is indicated by the bar. The experiment was performed in triplicate and a representative result is shown. Asterisks indicate significant differences compared to the wild-type attacker strain (NS, non significant; ***, p < 0.001).


A - B . Bacterial two - hybrid assay . BTH101 reporter cells producing the indicated proteins or domains fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on X - Gal indicator plates . The blue color of the colony reflects the interaction between the two proteins . TolB and Pal are two proteins known to interact but unrelated to the T6SS or the MltE proteins . The experiment was performed in triplicate and a representative result is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins or domains fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on X-Gal indicator plates. The blue color of the colony reflects the interaction between the two proteins. TolB and Pal are two proteins known to interact but unrelated to the T6SS or the MltE proteins. The experiment was performed in triplicate and a representative result is shown.


C . Co - immunoprecipitation assay . The solubilized lysates from 2 × 1010 E . coli K - 12 W3110 cells co - producing the indicated FLAG - tagged TssMP variants ( exported in the periplasm ) and VSV - G - tagged MltE protein ( Total , T ) were subjected to immune precipitation on anti - FLAG - coupled agarose beads . The immunoprecipitated material ( IP ) was subjected to 12 . 5 % - acrylamide SDS - PAGE and immunodetected with anti - FLAG ( upper panel , TssM domains ) and anti - VSV - G ( lower panel , MltE ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 E. coli K-12 W3110 cells co-producing the indicated FLAG-tagged TssMP variants (exported in the periplasm) and VSV-G-tagged MltE protein (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM domains) and anti-VSV-G (lower panel, MltE) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


A . Remazol brilliant blue assay . The absorbance of supernatants from the reaction containing purified and RBB - labelled E . coli peptidoglycan and the indicated protein ( 50 µg ) were measured at = 595 nm after incubation for 0 . 5 or 4 hours at 37 ° C . The results shown are the average and standard deviation from triplicate reactions ( n = 3 ) . Asterisks indicate significant differences compared to the buffer ( NS , non significant ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ) . The supernatant of the reaction after 4 hours of incubation is shown on bottom .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Remazol brilliant blue assay. The absorbance of supernatants from the reaction containing purified and RBB-labelled E. coli peptidoglycan and the indicated protein (50 µg) were measured at =595 nm after incubation for 0.5 or 4 hours at 37°C. The results shown are the average and standard deviation from triplicate reactions (n=3). Asterisks indicate significant differences compared to the buffer (NS, non significant; **, p < 0.01; ***, p < 0.001). The supernatant of the reaction after 4 hours of incubation is shown on bottom.


B . Peptidoglycan hydrolysis . The decrease of the absorbance of the M . luteus peptidoglycan suspension in presence of the indicated protein ( 50 µg ) was measured at = 600 nm at 37 ° C over time . The experiment was performed in triplicate and a representative result is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Peptidoglycan hydrolysis. The decrease of the absorbance of the M. luteus peptidoglycan suspension in presence of the indicated protein (50 µg) was measured at =600 nm at 37°C over time. The experiment was performed in triplicate and a representative result is shown.


A - B . Co - immunoprecipitation assay . The solubilized lysates from 2 × 1010 EAEC wild - type or ∆ mltE cells producing FLAG - tagged TssM ( FLTssM ) and / or HA - tagged TssJ ( TssJHA , panel A ) or TssL ( TssLHA , panel B ) ( Total , T ) were subjected to immune precipitation on anti - FLAG - coupled agarose beads . The immunoprecipitated material ( IP ) was subjected to 12 . 5 % - acrylamide SDS - PAGE and immunodetected with anti - FLAG ( upper panel , TssM ) and anti - HA ( lower panel , TssJ or TssL ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiments were performed in triplicate and a representative result is shown .

A-B. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 EAEC wild-type or ∆mltE cells producing FLAG-tagged TssM (FLTssM) and/or HA-tagged TssJ (TssJHA, panel A) or TssL (TssLHA, panel B) (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM) and anti-HA (lower panel, TssJ or TssL) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiments were performed in triplicate and a representative result is shown.


C . BS3 cross - linking assay . 2 × 109 cells of the indicated strain producing FLAG - tagged TssM ( with the exception of ∆ tssM cells ) were treated ( + ) or not ( - ) with the BS3 cross - linker agent . After the cross - linking reaction , cells were boiled in Laemmli buffer and total proteins were subjected to 7 % - acrylamide SDS - PAGE and immunodetected with anti - FLAG antibodies . The TssM protein ( FLTssM ) and its complexes ( * , TssM - TssJ ; * * , TssM - TssL ) are indicated on the right as well as the TssM dimer ( arrow ) . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BS3 cross-linking assay. 2×109 cells of the indicated strain producing FLAG-tagged TssM (with the exception of ∆tssM cells) were treated (+) or not (-) with the BS3 cross-linker agent. After the cross-linking reaction, cells were boiled in Laemmli buffer and total proteins were subjected to 7%-acrylamide SDS-PAGE and immunodetected with anti-FLAG antibodies. The TssM protein (FLTssM) and its complexes (*, TssM-TssJ; **, TssM-TssL) are indicated on the right as well as the TssM dimer (arrow). Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


D . Fluorescence microscopy . Recordings showing TssM localization using the chromosomally - encoded sfGFP - tssM fusion in wild - type ( WT ) or ΔmltE cells or ΔmltE cells producing the wild - type ( mltE + ) or catalytic variant ( mltEE64Q ) MltE protein . Scale bars are 1 μm . The experiment was performed in triplicate and a representative result is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fluorescence microscopy. Recordings showing TssM localization using the chromosomally-encoded sfGFP-tssM fusion in wild-type (WT) or ΔmltE cells or ΔmltE cells producing the wild-type (mltE+) or catalytic variant (mltEE64Q) MltE protein. Scale bars are 1 μm. The experiment was performed in triplicate and a representative result is shown.


B Ub chain interaction of the LUBEL - NZF and UBA2 domains . Immobilized GST - NZF or GST - UBA2 was incubated with Lys ( K ) 48 - , K 63 - linked , or linear di - Ub ( Ub2 ) chains and bound Ub chains were subjected to immunoblotting using anti - Ub antibody or a linear linkage - specific Ub antibody ( anti - linear Ub antibody ) . Loading of GST - fusion proteins was visualized by Ponceau S staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Ub chain interaction of the LUBEL-NZF and UBA2 domains. Immobilized GST-NZF or GST-UBA2 was incubated with Lys (K) 48-, K 63-linked, or linear di-Ub (Ub2) chains and bound Ub chains were subjected to immunoblotting using anti-Ub antibody or a linear linkage-specific Ub antibody (anti-linear Ub antibody). Loading of GST-fusion proteins was visualized by Ponceau S staining.


A In vitro ubiquitination assays of LUBEL - RBR - C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s , UbcD10 or Effete / UbcD1 . Reactions were terminated at indicated times and synthesized Ub chains were detected by immunoblotting using anti - linear Ub antibody . Total protein loading was visualized by Ponceau S staining . * : nonspecific band .

A In vitro ubiquitination assays of LUBEL-RBR-C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s, UbcD10 or Effete/UbcD1. Reactions were terminated at indicated times and synthesized Ub chains were detected by immunoblotting using anti-linear Ub antibody. Total protein loading was visualized by Ponceau S staining. * : nonspecific band.


B In vitro deubiquitination of Ub chains synthesized by HOIP - and LUBEL - RBR - C . Ub chains produced by RBR - C ( Human or Drosophila ) with UbcH7 , UbcD10 or Effete were incubated with a linear linkage - specific DUB , OTULIN ( WT or catalytically dead C129A ( C / A ) mutant ) or a Lys - linkage - specific DUB , vOTU . The DUB - treated samples were subjected to Coomassie staining or immunoblotting using anti - Ub antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro deubiquitination of Ub chains synthesized by HOIP- and LUBEL-RBR-C. Ub chains produced by RBR-C (Human or Drosophila) with UbcH7, UbcD10 or Effete were incubated with a linear linkage-specific DUB, OTULIN (WT or catalytically dead C129A (C/A) mutant) or a Lys-linkage-specific DUB, vOTU. The DUB-treated samples were subjected to Coomassie staining or immunoblotting using anti-Ub antibody.


C Thioester formation assay using Atto647 - labelled Ub . Ubiquitination assay using LUBEL - RBR - C WT ( left panel ) or C2704A ( right panel ) : lanes 1 / 2 Atto - Ub + E1 , lanes 3 / 4 + ATP , lanes 5 / 6 + E2 , lanes 7 / 8 + E3 , and lanes 9 / 10 + Ub - His6 . In the presence of N - terminally tagged Atto - Ub and C - terminally tagged Ub - His6 , only Ub2 product can be synthesized and longer chain formation is restricted . Samples are run without or with DTT in odd or even numbered lanes , respectively . The gels monitor the Atto - labelled Ub .

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

C Thioester formation assay using Atto647-labelled Ub. Ubiquitination assay using LUBEL-RBR-C WT (left panel) or C2704A (right panel): lanes 1/2 Atto-Ub + E1, lanes 3/4 + ATP, lanes 5/6 + E2, lanes 7/8 + E3, and lanes 9/10 + Ub-His6. In the presence of N-terminally tagged Atto-Ub and C-terminally tagged Ub-His6, only Ub2 product can be synthesized and longer chain formation is restricted. Samples are run without or with DTT in odd or even numbered lanes, respectively. The gels monitor the Atto-labelled Ub.


D The linear Ub chain formation activity of LUBEL - LDD mutants . Recombinant proteins of RBR - C WT , C2704A mutant , and LDD mutants ( R2754A and D2755A ) were assessed for their activity by in vitro ubiquitination assay . The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti - linear Ub antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The linear Ub chain formation activity of LUBEL-LDD mutants. Recombinant proteins of RBR-C WT, C2704A mutant, and LDD mutants (R2754A and D2755A) were assessed for their activity by in vitro ubiquitination assay. The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti-linear Ub antibody.


E Linear Ub chain formation by full - length LUBEL transient expression in insect cells . Full - length Myc - LUBEL was transiently expressed in Drosophila Schneider 2 ( S2 ) cells , and Myc - HOIP alone or with HA - HOIL - 1L in HEK293T cells . Total cell lysates ( TCL ) of control and transfected samples were incubated with immobilized GST - Linear - Tandem Ub binding entity ( Linear - TUBE ) containing three tandem repeats of ABIN - 1 - UBAN . Pulldown samples were blotted with anti - linear Ub antibody , while TCL were blotted with anti - Myc antibody for exogenous LUBEL and HOIP , anti - HA antibody for HOIL - 1L , and anti - Tubulin antibody for loading . Input of GST proteins was analyzed by Ponceau S staining . * : nonspecific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Linear Ub chain formation by full-length LUBEL transient expression in insect cells. Full-length Myc-LUBEL was transiently expressed in Drosophila Schneider 2 (S2) cells, and Myc-HOIP alone or with HA-HOIL-1L in HEK293T cells. Total cell lysates (TCL) of control and transfected samples were incubated with immobilized GST-Linear-Tandem Ub binding entity (Linear-TUBE) containing three tandem repeats of ABIN-1-UBAN. Pulldown samples were blotted with anti-linear Ub antibody, while TCL were blotted with anti-Myc antibody for exogenous LUBEL and HOIP, anti-HA antibody for HOIL-1L, and anti-Tubulin antibody for loading. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.


A Endogenous linear Ub chains enriched in total protein extracts ( input , lane 1 ) from adult w1118 ( w - ) flies by GST - Linear - TUBE ( lane 3 ) . The enriched samples were further treated with recombinant DUBs , vOTU ( lane 4 ) or OTULIN ( lane 5 ) . Ub chains were visualized by immunoblotting using anti - linear Ub antibody or anti - Ub antibody . Loading of GST proteins was analyzed by Ponceau S staining .

A Endogenous linear Ub chains enriched in total protein extracts (input, lane 1) from adult w1118 (w-) flies by GST-Linear-TUBE (lane 3). The enriched samples were further treated with recombinant DUBs, vOTU (lane 4) or OTULIN (lane 5). Ub chains were visualized by immunoblotting using anti-linear Ub antibody or anti-Ub antibody. Loading of GST proteins was analyzed by Ponceau S staining.


B Endogenous linear Ub chains in total cell lysate ( TCL ) of S2 cells expressing human Flag - OTULIN WT or catalytically inactive C129A mutant . TCLs enriched with GST - Linear - TUBE were examined by immunoblotting using anti - linear Ub antibody . Expression of OTULIN was analyzed by anti - Flag antibody and loading of TCL was detected by anti - Tubulin antibody . Input of GST proteins was analyzed by Ponceau S staining . * : nonspecific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Endogenous linear Ub chains in total cell lysate (TCL) of S2 cells expressing human Flag-OTULIN WT or catalytically inactive C129A mutant. TCLs enriched with GST-Linear-TUBE were examined by immunoblotting using anti-linear Ub antibody. Expression of OTULIN was analyzed by anti-Flag antibody and loading of TCL was detected by anti-Tubulin antibody. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.


C Mass spectrometry analysis using an identical sample as Figure 3B lane 6 . MS / MS spectra of the prototypic linear Ub chain peptide ( GGMQIFVK ) is shown .

O O O O O O O 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 Mass spectrometry analysis using an identical sample as Figure 3B lane 6. MS/MS spectra of the prototypic linear Ub chain peptide (GGMQIFVK) is shown.


C In vitro deubiquitination assay of dCYLD . dCYLD was incubated with all eight linkage types of Ub2 chains . dCYLD catalytically dead C284S mutant was incubated with K 63 - or linear Ub2 chains . All the proteins in the reactions were resolved on Coomassie - stained SDS - PAGE gel . * : nonspecific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro deubiquitination assay of dCYLD. dCYLD was incubated with all eight linkage types of Ub2 chains. dCYLD catalytically dead C284S mutant was incubated with K 63- or linear Ub2 chains. All the proteins in the reactions were resolved on Coomassie-stained SDS-PAGE gel. * : nonspecific band.


D Stabilization of linear Ub chains by dCYLD C284S mutant transient expression in S2 cells . Myc - tagged dCYLD WT and C284S were transfected in S2 cells and linear Ub chains were enriched with GST - Linear - TUBE and immunoblotted by anti - linear Ub or anti - Ub antibodies . Input of GST proteins was visualized by Ponceau S staining . Expression of Myc - dCYLD was examined by anti - Myc antibody , and anti - Tubulin antibody was used for the loading control of TCL .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Stabilization of linear Ub chains by dCYLD C284S mutant transient expression in S2 cells. Myc-tagged dCYLD WT and C284S were transfected in S2 cells and linear Ub chains were enriched with GST-Linear-TUBE and immunoblotted by anti-linear Ub or anti-Ub antibodies. Input of GST proteins was visualized by Ponceau S staining. Expression of Myc-dCYLD was examined by anti-Myc antibody, and anti-Tubulin antibody was used for the loading control of TCL.


E Linear Ub chains in dCYLD mutant flies . Endogenous levels of linear Ub chains in dCYLD mutant flies were compared with a control w - fly strain by performing GST - Linear - TUBE pulldown . Enriched linear Ub chains were immunoblotted with anti - linear Ub antibody . Input of GST proteins was visualized by Ponceau S staining . Tubulin was used for the loading control of the input .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Linear Ub chains in dCYLD mutant flies. Endogenous levels of linear Ub chains in dCYLD mutant flies were compared with a control w- fly strain by performing GST-Linear-TUBE pulldown. Enriched linear Ub chains were immunoblotted with anti-linear Ub antibody. Input of GST proteins was visualized by Ponceau S staining. Tubulin was used for the loading control of the input.


B Linear Ub chains in w - and LUBEL mutant fly lysates . w - , CC / SS and delR2 flies were analyzed for the level of endogenous linear Ub chains by enriching the linear ubiquitin chains by GST - Linear - TUBE . Pulldown samples were immunoblotted by anti - linear Ub antibody , while GST - proteins were analyzed by Ponceau S staining . Anti - Tubulin antibody was used for the loading control of the input . * : nonspecific band .

B Linear Ub chains in w- and LUBEL mutant fly lysates. w-, CC/SS and delR2 flies were analyzed for the level of endogenous linear Ub chains by enriching the linear ubiquitin chains by GST-Linear-TUBE. Pulldown samples were immunoblotted by anti-linear Ub antibody, while GST-proteins were analyzed by Ponceau S staining. Anti-Tubulin antibody was used for the loading control of the input. * : nonspecific band.


C The lifespans of female w - and catalytically dead LUBEL mutant flies ( two clones of CC / SS , indicated as # 1 and # 2 , and delR2 ) . Survival of three independent cohorts with approximately 80 flies each was monitored over time . Median survival time ( days ) : w - = 78 , CC / SS # 1 = 68 , CC / SS # 2 = 65 , and delR2 = 71 . Total sample sizes are as follow ; w - = 215 , CC / SS # 1 = 242 , CC / SS # 2 = 233 , delR2 = 273 . Log - rank ( Mantel - cox ) test between fly lines : w - and CC / SS # 1 > 0 . 0001 , w - and CC / SS # 2 > 0 . 0001 , w - and delR2 > 0 . 0001 , CC / SS # 1 and CC / SS # 2 = 0 . 0007 , CC / SS # 1 and delR2 = not significant , CC / SS # 2 and delR2 = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The lifespans of female w- and catalytically dead LUBEL mutant flies (two clones of CC/SS, indicated as #1 and #2, and delR2). Survival of three independent cohorts with approximately 80 flies each was monitored over time. Median survival time (days): w- = 78, CC/SS #1 = 68, CC/SS #2 = 65, and delR2 = 71. Total sample sizes are as follow; w- = 215, CC/SS #1 = 242, CC/SS #2 = 233, delR2 = 273. Log-rank (Mantel-cox) test between fly lines: w- and CC/SS #1 > 0.0001, w- and CC/SS #2 > 0.0001, w- and delR2 > 0.0001, CC/SS #1 and CC/SS #2 = 0.0007, CC/SS #1 and delR2 = not significant, CC/SS #2 and delR2 = not significant.


D Histological analysis of muscle in LUBEL mutant flies . Hematoxylin and eosin ( H & amp ; amp ; E ) ( top panels ) and actin immunofluorescent staining ( Actin - IF ) ( bottom panels ) of thorax muscles from young ( Day 3 ) or aged ( Day 60 ) w - or CC / SS female flies . 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 O O O O O O O O O O O O O O O O O O O

D Histological analysis of muscle in LUBEL mutant flies. Hematoxylin and eosin (H&amp;E) (top panels) and actin immunofluorescent staining (Actin-IF) (bottom panels) of thorax muscles from young (Day 3) or aged (Day 60) w- or CC/SS female flies. Scale bars: 50 μm.


A The level of poly - Ub chains in w - flies after heat shock . w - male flies were incubated in a 36 ° C water bath for indicated times and the levels of poly - Ub chains in total protein extracts were compared by Ub using antibodies against linear , K 48 - , and K 63 - linked Ub chains . Ponceau S staining was used to visualize the protein loading .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The level of poly-Ub chains in w- flies after heat shock. w- male flies were incubated in a 36 °C water bath for indicated times and the levels of poly-Ub chains in total protein extracts were compared by Ub using antibodies against linear, K 48-, and K 63-linked Ub chains. Ponceau S staining was used to visualize the protein loading.


B Survival of catalytically dead LUBEL mutant flies upon heat shock . Young adult flies ( 15 males and 15 females ) were incubated in a 36 ° C water bath and immobilized flies were counted over the time indicated . Median survival time ( hours ) : w - = 9 . 25 , CC / SS # 1 = 5 . 5 , CC / SS # 2 = 5 , delR2 = 5 . 75 , CC / SS # 1 / + = 8 . 5 , CC / SS # 2 / + = 10 , delR2 / + = 8 . p - values calculated by Gehan - Breslow - Wilcoxon test between fly lines : w - and CC / SS # 1 , CC / SS # 2 , or delR2 > 0 . 0001 , w - and CC / SS # 1 / + = 0 . 0134 , w - and CC / SS # 2 / + = ns , w - and delR2 / + = 0 . 0002 , CC / SS # 1 and CC / SS # 1 / + > 0 . 0001 , CC / SS # 2 and CC / SS # 2 / + > 0 . 0001 , delR2 and delR2 / + > 0 . 0001 . Representative data are shown from six independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Survival of catalytically dead LUBEL mutant flies upon heat shock. Young adult flies (15 males and 15 females) were incubated in a 36 °C water bath and immobilized flies were counted over the time indicated. Median survival time (hours): w- = 9.25, CC/SS #1 = 5.5, CC/SS #2 = 5, delR2 = 5.75, CC/SS #1/+ = 8.5, CC/SS #2/+ = 10, delR2/+ = 8. p-values calculated by Gehan-Breslow-Wilcoxon test between fly lines: w- and CC/SS #1, CC/SS#2, or delR2 > 0.0001, w- and CC/SS #1/+ = 0.0134, w- and CC/SS #2/+ = ns, w- and delR2/+ = 0.0002, CC/SS #1 and CC/SS #1/+ > 0.0001, CC/SS #2 and CC/SS #2/+ > 0.0001, delR2 and delR2/+ > 0.0001. Representative data are shown from six independent experiments.


C Survival of whole body or muscle - specific LUBEL knockdown flies upon heat shock . shRNA - based knockdown ( KD ) of LUBEL were driven by Tub - Gal4 or Mef2 - Gal4 flies . Control fly lines ( Tub - Gal4 / + and Mef2 - Gal4 / + ) were used to compare with the KD flies . 15 males and 15 females per each line were used in this assay . Median survival time ( hours ) : Tub - Gal4 / + = 7 , Tub - Gal4 > LUBEL = 3 . 5 , Mef2 - Gal4 / + = 7 . 5 , Mef2 - Gal4 > LUBEL = 7 . p - values calculated by Gehan - Breslow - Wilcoxon test : Tub - Gal4 < 0 . 0001 ( * * * * ) , Mef2 - Gal4 = 0 . 0029 ( * * ) . Representative data are shown from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Survival of whole body or muscle-specific LUBEL knockdown flies upon heat shock. shRNA-based knockdown (KD) of LUBEL were driven by Tub-Gal4 or Mef2-Gal4 flies. Control fly lines (Tub-Gal4/+ and Mef2-Gal4/+) were used to compare with the KD flies. 15 males and 15 females per each line were used in this assay. Median survival time (hours): Tub-Gal4/+ = 7, Tub-Gal4>LUBEL = 3.5, Mef2-Gal4/+ = 7.5, Mef2-Gal4>LUBEL= 7. p-values calculated by Gehan-Breslow-Wilcoxon test: Tub-Gal4 <0.0001 (****), Mef2-Gal4 = 0.0029 (**). Representative data are shown from three independent experiments.


D Heat - induced mRNA expression of HSP70 . w - , CC / SS # 2 and delR2 flies were heat treated for 60 min and recovered for 1 hour , and mRNA HSP70 was quantified by qPCR . Rp49 was used as a reference and w - untreated sample was used as calibrator to calculate the expression ratio . Data are presented as meanSD ( * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) . Representative data are shown from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heat-induced mRNA expression of HSP70. w-, CC/SS #2 and delR2 flies were heat treated for 60 min and recovered for 1 hour, and mRNA HSP70 was quantified by qPCR. Rp49 was used as a reference and w- untreated sample was used as calibrator to calculate the expression ratio. Data are presented as meanSD (*<0.01, ***<0.001, ****<0.0001). Representative data are shown from three independent experiments.


( b ) Levels of spermidine in the heads of wild - type flies fed with food supplemented by 1 or 5 mM spermidine ( Spd1mM + or Spd5mM + , respectively ) or food with no spermidine ( Spd − ) . Data are shown normalized to spermidine levels of 1 - d - old ( 1 d ) Spd − flies ( n = 4 independent experiments ; F = 14 . 08 for Spd − flies , F = 5 . 30 for Spd1mM + flies , F = 2 . 62 for Spd5mM + flies ; one - way - ANOVA with Bonferroni correction ) . 15 d , 15 d old ; 30 d , 30 d old .

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 B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR 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 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) Levels of spermidine in the heads of wild-type flies fed with food supplemented by 1 or 5 mM spermidine (Spd1mM+ or Spd5mM+, respectively) or food with no spermidine (Spd−). Data are shown normalized to spermidine levels of 1-d-old (1 d) Spd− flies (n = 4 independent experiments; F = 14.08 for Spd− flies, F = 5.30 for Spd1mM+ flies, F = 2.62 for Spd5mM+ flies; one-way-ANOVA with Bonferroni correction). 15 d, 15 d old; 30 d, 30 d old.


( c ) Levels of putrescine in the heads of aged Spd1mM + or Spd5mM + flies compared with Spd − flies . Data are shown normalized to putrescine levels of 1 - d - old Spd − flies ( n = 4 independent experiments ; F = 24 . 31 for Spd - flies , F = 0 . 22 for Spd1mM + flies , F = 6 . 67 for Spd5mM + flies ; one - way - ANOVA with Bonferroni correction ) . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; ns indicates not significant , P > 0 . 05 . Data are presented as mean ± s . e . m .

O O O O O B-MEASURED_VAR 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-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Levels of putrescine in the heads of aged Spd1mM+ or Spd5mM+ flies compared with Spd− flies. Data are shown normalized to putrescine levels of 1-d-old Spd− flies (n = 4 independent experiments; F = 24.31 for Spd- flies, F = 0.22 for Spd1mM+ flies, F = 6.67 for Spd5mM+ flies; one-way-ANOVA with Bonferroni correction). *P 0.05; **P 0.01; ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.


( a ) Aversive associative memory performance at 3 min after training ( STM ) of 3 - d - old ( 3 d ) Spd1mM + and Spd5mM + flies in comparison to Spd − flies ( n = 8 - 9 independent experiments , F = 2 . 28 , one - way - ANOVA with Bonferroni correction ) .

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

(a) Aversive associative memory performance at 3 min after training (STM) of 3-d-old (3 d) Spd1mM+ and Spd5mM+ flies in comparison to Spd− flies (n = 8-9 independent experiments, F = 2.28, one-way-ANOVA with Bonferroni correction).


( b ) Aversive associative memory performance at 3 h after training ( ITM ) , ARM and ASM of 3 - d - old Spd1mM + and Spd5mM + compared with Spd − flies ( n = 8 - 9 independent experiments ; F = 2 . 05 for ITM , F = 0 . 02 for ARM , F = 1 . 74 for ASM ; one - way ANOVA with Bonferroni correction ) .

O 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 B-CONTROLLED_VAR 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

(b) Aversive associative memory performance at 3 h after training (ITM), ARM and ASM of 3-d-old Spd1mM+ and Spd5mM+ compared with Spd− flies (n = 8-9 independent experiments; F = 2.05 for ITM, F = 0.02 for ARM, F = 1.74 for ASM; one-way ANOVA with Bonferroni correction).


( c ) STM of 30 - d - old ( 30 d ) Spd1mM + and Spd5mM + and Spd − flies ( n = 8 - 9 independent experiments , F = 14 . 48 , one - way ANOVA with Bonferroni correction ) .

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

(c) STM of 30-d-old (30 d) Spd1mM+ and Spd5mM+ and Spd− flies (n = 8-9 independent experiments, F = 14.48, one-way ANOVA with Bonferroni correction).


( d ) ITM , ARM and ASM of 30 - d - old Spd1mM + and Spd5mM + compared with Spd − flies ( n = 7 - 9 independent experiments ; F = 43 . 26 for ITM , F = 1 . 74 for ARM , F = 26 . 12 for ASM ; one - way ANOVA with Bonferroni correction ) .

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

(d) ITM, ARM and ASM of 30-d-old Spd1mM+ and Spd5mM+ compared with Spd− flies (n = 7-9 independent experiments; F = 43.26 for ITM, F = 1.74 for ARM, F = 26.12 for ASM; one-way ANOVA with Bonferroni correction).


( e ) Spermidine feeding for 10 d before measuring memory was sufficient to suppress AMI in aged ( 30 d old ) flies ( n = 9 - 11 independent experiments , F = 12 . 32 , one - way ANOVA with Bonferroni correction ) .

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

(e) Spermidine feeding for 10 d before measuring memory was sufficient to suppress AMI in aged (30 d old) flies (n = 9-11 independent experiments, F = 12.32, one-way ANOVA with Bonferroni correction).


( f , g ) Spermidine feeding on days 21 - 30 resulted in a significant increase in the levels of spermidine and putrescine in 30 - d - old flies ( n = 3 independent experiments , F = 32 . 96 ( f ) and 64 . 77 ( g ) , one - way ANOVA with Bonferroni correction ) . The feeding regime of the flies is presented beneath the graphs , with + and − indicating food with or without spermidine . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; ns indicates not significant , P > 0 . 05 . Data are presented as mean ± s . e . m .

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

(f,g) Spermidine feeding on days 21-30 resulted in a significant increase in the levels of spermidine and putrescine in 30-d-old flies (n = 3 independent experiments, F = 32.96 (f) and 64.77 (g), one-way ANOVA with Bonferroni correction). The feeding regime of the flies is presented beneath the graphs, with + and − indicating food with or without spermidine. *P 0.05, **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.


( a ) Expression of GCaMP3 . 0 in the mushroom body of an individual fly , focused on the horizontal lobes . The red line indicates a region of interest used to determine changes in fluorescence emission . Scale bar represents 50 μm . ( b ) False color - coded image of Ca2 + activity in the horizontal mushroom body lobes shown in a . Warm colors indicate high levels , cold colors indicate low levels or no Ca2 + activity . The numbers indicate changes in fluorescence ΔF ( % ) .

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

(a) Expression of GCaMP3.0 in the mushroom body of an individual fly, focused on the horizontal lobes. The red line indicates a region of interest used to determine changes in fluorescence emission. Scale bar represents 50 μm. (b) False color-coded image of Ca2+ activity in the horizontal mushroom body lobes shown in a. Warm colors indicate high levels, cold colors indicate low levels or no Ca2+ activity. The numbers indicate changes in fluorescence ΔF (%).


( c , d ) Time course of Ca2 + increase in horizontal mushroom body lobes of 3 - d - old and 30 - d - old Spd − flies evoked by the odors 4 - methyl - cyclohexanol ( MCH ) or 3 - octanol ( OCT ) in comparison with the diluent , mineral oil ( n = 5 flies , non - parametric Mann - Whitney U test found no substantial difference between the Ca2 + increase of 3 - d - old and 30 - d - old Spd − flies evoked by the odors ) . The gray bars indicate the duration of the odor stimuli . Data are presented as mean ± s . e . m .

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

(c,d) Time course of Ca2+ increase in horizontal mushroom body lobes of 3-d-old and 30-d-old Spd− flies evoked by the odors 4-methyl-cyclohexanol (MCH) or 3-octanol (OCT) in comparison with the diluent, mineral oil (n = 5 flies, non-parametric Mann-Whitney U test found no substantial difference between the Ca2+ increase of 3-d-old and 30-d-old Spd− flies evoked by the odors). The gray bars indicate the duration of the odor stimuli. Data are presented as mean ± s.e.m.


( a ) Quantification of Atg8a proteins levels from head extracts normalized to α - tubulin ( n = 17 or 8 - 9 technical replicates from two independent biological aging replicates for all data , F = 7 . 52 , one - way - ANOVA with Tukey post - test ) . The full - length blot is shown in Supplementary Figure 9a .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of Atg8a proteins levels from head extracts normalized to α-tubulin (n = 17 or 8-9 technical replicates from two independent biological aging replicates for all data, F = 7.52, one-way-ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9a.


( b ) Western blot analysis of poly - ubiquitinated proteins ( SDS - soluble protein fraction ) from head extracts from Spd − ( 3 and 30 d old ) flies compared with Spd5mM + ( 3 and 30 d old ) flies . Right , quantification of poly - ubiquitinated proteins levels normalized to α - tubulin ( n = 10 or 5 technical replicates from two independent biological aging replicates for all data , F = 6 . 32 , one - way ANOVA with Tukey post - test ) . The full - length blot is shown in Supplementary Figure 9b .

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 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

(b) Western blot analysis of poly-ubiquitinated proteins (SDS-soluble protein fraction) from head extracts from Spd− (3 and 30 d old) flies compared with Spd5mM+ (3 and 30 d old) flies. Right, quantification of poly-ubiquitinated proteins levels normalized to α-tubulin (n = 10 or 5 technical replicates from two independent biological aging replicates for all data, F = 6.32, one-way ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9b.


( c - f ) Adult brains of 3 - d - old and 30 - d - oldSpd − flies , as well as 3 - d - old and 30 - d - oldSpd5mM + fliesimmunostained for ref ( 2 ) P . Scale bar represents 25 μm . ( g ) Quantification of ref ( 2 ) P intensity in the central brain region normalized to 3 - d - oldSpd − ( n = 12 - 13 independent brains for all data , F = 26 . 58 , one - way ANOVA with Bonferroni correction ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; P > 0 . 05 . Data are presented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f) Adult brains of 3-d-old and 30-d-oldSpd− flies, as well as 3-d-old and 30-d-oldSpd5mM+ fliesimmunostained for ref(2)P. Scale bar represents 25 μm. (g) Quantification of ref(2)P intensity in the central brain region normalized to 3-d-oldSpd− (n = 12-13 independent brains for all data, F = 26.58, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; P > 0.05. Data are presented as mean ± s.e.m.


( a ) Aversive associative memory performance 3 min after training ( STM ) was markedly reduced in 3 - d - old Atg7 − / − flies ( Atg7d14 / Atg7d77 ) compared with Atg7 − / + flies ( Atg7d14 / CG5335d30 ) and wild - type flies ( Atg7 + / + ) ( n = 8 - 11 independent experiments , F = 25 . 82 , one - way ANOVA with Bonferroni correction ) . The CG5335d30 line harbors an Atg7 + chromosome related to Atg7d14 and Atg7d77 that serves as a genetic background control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Aversive associative memory performance 3 min after training (STM) was markedly reduced in 3-d-old Atg7−/− flies (Atg7d14/Atg7d77) compared with Atg7−/+ flies (Atg7d14/CG5335d30) and wild-type flies (Atg7+/+) (n = 8-11 independent experiments, F = 25.82, one-way ANOVA with Bonferroni correction). The CG5335d30 line harbors an Atg7+ chromosome related to Atg7d14 and Atg7d77 that serves as a genetic background control.


( b ) Olfactory learning was disrupted in 3 - d - old female flies homozygous for Atg8a hypomorph ( Atg8a − / − or Atg8aEP362 / Atg8aEP362 ) ( n = 8 - 9 independent experiments , F = 22 . 8 , one - way ANOVA with Bonferroni correction ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Olfactory learning was disrupted in 3-d-old female flies homozygous for Atg8a hypomorph (Atg8a−/− or Atg8aEP362/Atg8aEP362) (n = 8-9 independent experiments, F = 22.8, one-way ANOVA with Bonferroni correction).


( c ) STM is severely impaired in 20 - d - old Atg7 − / − ( Atg7d14 / Atg7d77 ) flies and 20 - d - old Atg7 − / + heterozygous ( Atg7d14 / CG5335d30 ) when compared to age - matched wild - type flies ( n = 8 - 9 independent experiments , F = 18 . 02 , one - way ANOVA with Bonferroni correction ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) STM is severely impaired in 20-d-old Atg7−/− (Atg7d14/Atg7d77) flies and 20-d-old Atg7−/+ heterozygous (Atg7d14/CG5335d30) when compared to age-matched wild-type flies (n = 8-9 independent experiments, F = 18.02, one-way ANOVA with Bonferroni correction).


( d ) Similarly , STM was markedly decreased in 20 - d - old Atg8a − / − ( Atg8aEP362 / Atg8aEP362 ) mutant female flies compared with control flies ( Atg8a + / + ) without any effect by spermidine feeding ( n = 8 - 9 independent experiments , F = 68 . 43 , one - way ANOVA with Bonferroni correction ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; ns indicates not significant , P > 0 . 05 . Data are presented as mean ± s . e . m . The aversive olfactory avoidance and shock reactivity of different mutant are shown in Supplementary Table 5 .

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

(d) Similarly, STM was markedly decreased in 20-d-old Atg8a−/− (Atg8aEP362/Atg8aEP362) mutant female flies compared with control flies (Atg8a+/+) without any effect by spermidine feeding (n = 8-9 independent experiments, F = 68.43, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m. The aversive olfactory avoidance and shock reactivity of different mutant are shown in Supplementary Table 5.


( a ) Aversive associative memory performance in 3 min after training ( STM ) in 3 - d - old female flies with pan - neuronal expression of UAS - Odc - 1 ( n = 7 independent experiments , F = 0 . 04 , one - way ANOVA with Bonferroni correction ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Aversive associative memory performance in 3 min after training (STM) in 3-d-old female flies with pan-neuronal expression of UAS-Odc-1 (n = 7 independent experiments, F = 0.04, one-way ANOVA with Bonferroni correction).


( b ) Aversive associative memory performance in 3 h after training ( ITM ) , ARM and ASM of 3 - d - old appl - driven UAS - Odc - 1 female flies compared with control flies ( n = 7 independent experiments ; F = 1 . 95 for ITM , F = 0 . 10 for ARM , F = 1 . 05 for ASM ; one - way ANOVA with Bonferroni correction ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Aversive associative memory performance in 3 h after training (ITM), ARM and ASM of 3-d-old appl-driven UAS-Odc-1 female flies compared with control flies (n = 7 independent experiments; F = 1.95 for ITM, F = 0.10 for ARM, F = 1.05 for ASM; one-way ANOVA with Bonferroni correction).


( c ) Pan - neuronal expression of the UAS - Odc - 1 in a wild - type background suppressed AMI in 30 - d - old female flies ( n = 7 independent experiments , F = 21 . 35 , one - way - ANOVA with Bonferroni correction ) .

(c) Pan-neuronal expression of the UAS-Odc-1 in a wild-type background suppressed AMI in 30-d-old female flies (n = 7 independent experiments, F = 21.35, one-way-ANOVA with Bonferroni correction).


( d ) ITM , ARM and ASM of 30 - d - old female flies with pan - neuronal expression of UAS - Odc - 1 compared with their genetic controls ( n = 7 - 8 independent experiments ; F = 17 . 49 for ITM , F = 1 . 58 for ARM , F = 24 . 61 for ASM ; one - way ANOVA with Bonferroni correction ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ITM, ARM and ASM of 30-d-old female flies with pan-neuronal expression of UAS-Odc-1 compared with their genetic controls (n = 7-8 independent experiments; F = 17.49 for ITM, F = 1.58 for ARM, F = 24.61 for ASM; one-way ANOVA with Bonferroni correction).


( e ) Expressing Odc - 1 in just the mushroom body was sufficient to protect from STM decline in 30 - d - old flies ( n = 9 - 12 independent experiments , F = 9 . 25 , one - way ANOVA with Bonferroni correction ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expressing Odc-1 in just the mushroom body was sufficient to protect from STM decline in 30-d-old flies (n = 9-12 independent experiments, F = 9.25, one-way ANOVA with Bonferroni correction).


( f ) ITM , ARM and ASM of 30 - d - old flies expressing mushroom body - specific Odc - 1 compared with their genetic controls ( n = 8 - 9 independent experiments ; F = 11 . 73 for ITM , F = 0 . 50 for ARM , F = 16 . 6 for ASM ; one - way ANOVA with Bonferroni correction ) . * * P 0 . 01 , * * * P 0 . 001 ; ns indicates not significant , P > 0 . 05 . Data are presented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ITM, ARM and ASM of 30-d-old flies expressing mushroom body-specific Odc-1 compared with their genetic controls (n = 8-9 independent experiments; F = 11.73 for ITM, F = 0.50 for ARM, F = 16.6 for ASM; one-way ANOVA with Bonferroni correction). **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.


Neuroblastoma PDX and SK - N - BE ( 2 ) c cells treated with IBL inhibitors at indicated concentrations for 48 hours . pAkt ( at Ser473 ( A ) and Thr308 ( B ) sites ) levels in LU - NB - 3 and SK - N - BE ( 2 ) c cells determined by western blotting . Total Akt levels were used as loading control .

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

Neuroblastoma PDX and SK-N-BE(2)c cells treated with IBL inhibitors at indicated concentrations for 48 hours. pAkt (at Ser473 (A) and Thr308 (B) sites) levels in LU-NB-3 and SK-N-BE(2)c cells determined by western blotting. Total Akt levels were used as loading control.


Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours . C . p - p70S6K and p - p85S6K levels in LU - NB - 3 cells determined by western blotting . Actin , p70S6K , and p85S6K levels were used as loading controls .

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

Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours. C. p-p70S6K and p-p85S6K levels in LU-NB-3 cells determined by western blotting. Actin, p70S6K, and p85S6K levels were used as loading controls.


D . Brightfield photomicrographs of LU - NB - 3 and SK - N - BE ( 2 ) c cells treated with 0 . 36μM IBL - 202 or 0 . 05 μM IBL - 301 . Scale bars represent 100 μm ( LU - NB - 3 ) or 200 μm ( SK - N - BE ( 2 ) c ) . Arrows indicate neurite outgrowths and asterisks indicate where inserts are magnified . IBL - 301 treated cells were stained for Tuj1 . DAPI was used to visualize nuclei . E . Quantification of neurite outgrowth presented as number of neurites / cell in LU - NB - 3 PDX and SK - N - BE ( 2 ) cells treated with IBL - 301 . For LU - NB - 3 PDX cells , representative areas ( n = 2 ) were used and n = 344 and n = 240 cells / condition for CTRL and IBL - 301 , respectively , were counted . For SK - N - BE ( 2 ) c cells , representative areas ( n = 2 and n = 3 for CTRL and IBL - 301 respectively ) were used and n = 141 and n = 130 cells / condition for CTRL and IBL - 301 , respectively , were counted . Statistical significance was determined by two - sided student ' s t - tes . P = 0 . 003 for LU - NB - 3 and p = 0 . 08 for SK - N - BE ( 2 ) c .

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

D. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 0.36μM IBL-202 or 0.05 μM IBL-301. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). Arrows indicate neurite outgrowths and asterisks indicate where inserts are magnified. IBL-301 treated cells were stained for Tuj1. DAPI was used to visualize nuclei. E. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2) cells treated with IBL-301. For LU-NB-3 PDX cells, representative areas (n=2) were used and n=344 and n=240 cells/condition for CTRL and IBL-301, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=2 and n=3 for CTRL and IBL-301 respectively) were used and n=141 and n=130 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-tes. P=0.003 for LU-NB-3 and p=0.08 for SK-N-BE(2)c.


Neuroblastoma PDX and SK - N - BE ( 2 ) c cells treated with IBL inhibitors at indicated concentrations for 48 hours . F . Gap43 protein levels in LU - NB - 3 and SK - N - BE ( 2 ) c cells determined by western blotting . SDHA levels were used as loading control .

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

Neuroblastoma PDX and SK-N-BE(2)c cells treated with IBL inhibitors at indicated concentrations for 48 hours. F. Gap43 protein levels in LU-NB-3 and SK-N-BE(2)c cells determined by western blotting. SDHA levels were used as loading control.


Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours . G . N - Myc levels in LU - NB - 2 and LU - NB - 3 cells determined by western blotting . SDHA levels were used as loading control .

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

Neuroblastoma PDX cells treated with IBL inhibitors at indicated concentrations for 48 hours. G. N-Myc levels in LU-NB-2 and LU-NB-3 cells determined by western blotting. SDHA levels were used as loading control.


A . Viability IBL - 202 or IBL - 301 treated cells determined by CellTiterGlo .

A. Viability IBL-202 or IBL-301 treated cells determined by CellTiterGlo.


B - C . Cell cycle distribution of LU - NB - 3 cells determined by flow cytometry . Bars show mean values from two independent experiments .

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

B-C. Cell cycle distribution of LU-NB-3 cells determined by flow cytometry. Bars show mean values from two independent experiments.


D . Cleaved caspase 3 ( Cl Casp3 ) levels in LU - NB - 3 cells determined by western blotting . SDHA levels were used as loading control .

O O O O O O 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. Cleaved caspase 3 (Cl Casp3) levels in LU-NB-3 cells determined by western blotting. SDHA levels were used as loading control.


E . Flow cytometry analyses of Annexin V and PI stainings following treatment with 360 nM IBL - 202 or 50 nM IBL - 301 . F . Quantification of live and dead cells from the AnnexinV / PI stainings . Dead cells = PI positive , Live cells = PI negative . Dot plots from two independent experiments and error bars represent SEM .

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

E. Flow cytometry analyses of Annexin V and PI stainings following treatment with 360 nM IBL-202 or 50 nM IBL-301. F. Quantification of live and dead cells from the AnnexinV/PI stainings. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM.


Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours A . Expression of pAkt ( Ser473 ) in LU - NB - 3 and SK - N - BE ( 2 ) c cells . Total Akt and actin were used as loading controls . B . Expression of pAkt ( Thr308 ) in LU - NB - 3 and SK - N - BE ( 2 ) c cells . Total Akt and actin were used as loading controls .

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

Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours A. Expression of pAkt(Ser473) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls. B. Expression of pAkt(Thr308) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls.


Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours C . Brightfield photomicrographs of LU - NB - 3 and SK - N - BE ( 2 ) c cells treated with 50 nM IBL - 302 . Scale bars represent 100 μm ( LU - NB - 3 ) or 200 μm ( SK - N - BE ( 2 ) c ) . D . Quantification of neurite outgrowth presented as number of neurites / cell in LU - NB - 3 PDX and SK - N - BE ( 2 ) c cells treated with IBL - 302 . For LU - NB - 3 PDX cells , representative areas ( n = 4 ) were used and n = 460 and n = 216 cells / condition for CTRL and IBL - 302 , respectively , were counted . For SK - N - BE ( 2 ) c cells , representative areas ( n = 5 and n = 7 for CTRL and IBL - 302 respectively ) were used and n = 124 and n = 260 cells / condition for CTRL and IBL - 301 , respectively , were counted . Statistical significance was determined by two - sided student ' s t - test . p = 0 . 009 for LU - NB - 3 and p = 0 . 0003 for SK - N - BE ( 2 ) c .

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

Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours C. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 50 nM IBL-302. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). D. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2)c cells treated with IBL-302. For LU-NB-3 PDX cells, representative areas (n=4) were used and n=460 and n=216 cells/condition for CTRL and IBL-302, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=5 and n=7 for CTRL and IBL-302 respectively) were used and n=124 and n=260 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-test. p=0.009 for LU-NB-3 and p=0.0003 for SK-N-BE(2)c.