Figure 1 ( A ) Representative image of a typical young WT cortico - hippocampal slice ( 350 µm ) from co - culture ( 14 DIV ) stained with DAPI ( gray ) to visualize nuclei ( left ) . Dentate gyrus ( DG ) , cornus ammonis 1 ( CA1 ) and cortical ( CTX ) regions are indicated . An example of the co - culture dish is shown on the right . Co - culture includes 2 young WT ( * , young co - culture ) and 2 old APPPS1 ( + , old co - culture ) brain slices . ( B ) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone ( young alone , old alone ) or in co - culture ( young co - culture , old co - culture ) at 7 days in vitro ( DIV ) stained with propidium iodide ( PI , red ) reveals reduced cell viability in old brain slices compared to young . ( C ) Quantitative analysis of PI positive cells in young WT and old APPPS1 slices cultured alone ( white and black bars , respectively ) or in co - culture ( grey and white squared bars , respectively ) at 7 and 14 DIV . The values are expressed as percentages of PI positive cells from the total number of DAPI positive cells . The values represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( n . s . = not significant ; * * * P < 0 . 001 ) . ( D - F ) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone or in co - culture at 7 DIV and immunostained using neuronal ( NeuN ) , astrocytic ( GFAP ) and microglial ( CD68 ) markers ( green ) reveals reduced NeuN and GFAP and increased CD68 immunosignal . Scale bar : 75

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

Figure 1(A) Representative image of a typical young WT cortico-hippocampal slice (350 µm) from co-culture (14 DIV) stained with DAPI (gray) to visualize nuclei (left). Dentate gyrus (DG), cornus ammonis 1 (CA1) and cortical (CTX) regions are indicated. An example of the co-culture dish is shown on the right. Co-culture includes 2 young WT (*, young co-culture) and 2 old APPPS1 (+, old co-culture) brain slices.(B) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone (young alone, old alone) or in co-culture (young co-culture, old co-culture) at 7 days in vitro (DIV) stained with propidium iodide (PI, red) reveals reduced cell viability in old brain slices compared to young.(C) Quantitative analysis of PI positive cells in young WT and old APPPS1 slices cultured alone (white and black bars, respectively) or in co-culture (grey and white squared bars, respectively) at 7 and 14 DIV. The values are expressed as percentages of PI positive cells from the total number of DAPI positive cells. The values represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (n.s.= not significant; ***P < 0.001).(D-F) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone or in co-culture at 7 DIV and immunostained using neuronal (NeuN), astrocytic (GFAP) and microglial (CD68) markers (green) reveals reduced NeuN and GFAP and increased CD68 immunosignal. Scale bar: 75 µm


Figure 2 ( A ) Immunofluorescence analysis of the old APPPS1 slice in co - culture using antibody 6E10 to detect amyloid plaques ( green ) and Thiazine Red ( TR , red ) to visualize fibrillar amyloid cores . At 7 DIV , the majority of amyloid plaques contain 6E10 and TR positive dense core surrounded by 6E10 positive halo of diffuse Aβ . In contrast , at 14 DIV , the majority of amyloid plaques are core - only plaques co - stained with 6E10 and TR without detectable 6E10 positive halo . Lower panels are higher magnification images of boxed regions . Scale bars : 50 µm . ( B ) Quantitative analysis of core - only plaques in old APPPS1 slices cultured alone ( white bars ) or in co - culture ( gray bars ) at 7 and 14 DIV reveals an increased number of core - only plaques upon co - culturing of old APPPS1 brain slices together with young WT 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 ) . ( C ) Western blot analysis of Aβ levels ( triplicates ) in the old APPPS1 tissue cultured alone or in co - culture with the young WT ( 14 DIV ) using 2D8 antibody . ( D ) Quantification of Western blot signals reveals decreased levels of Aβ upon co - culturing compared to old tissue cultured alone . The values are normalized to the levels of Aβ in the old tissue cultured alone and represent mean ± SEM from 2 independent experiments , each experiment was performed in triplicates ( * * P < 0 . 01 ) .

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

Figure 2(A) Immunofluorescence analysis of the old APPPS1 slice in co-culture using antibody 6E10 to detect amyloid plaques (green) and Thiazine Red (TR, red) to visualize fibrillar amyloid cores. At 7 DIV, the majority of amyloid plaques contain 6E10 and TR positive dense core surrounded by 6E10 positive halo of diffuse Aβ. In contrast, at 14 DIV, the majority of amyloid plaques are core-only plaques co-stained with 6E10 and TR without detectable 6E10 positive halo. Lower panels are higher magnification images of boxed regions. Scale bars: 50 µm.(B) Quantitative analysis of core-only plaques in old APPPS1 slices cultured alone (white bars) or in co-culture (gray bars) at 7 and 14 DIV reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young WT 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).(C) Western blot analysis of Aβ levels (triplicates) in the old APPPS1 tissue cultured alone or in co-culture with the young WT (14 DIV) using 2D8 antibody.(D) Quantification of Western blot signals reveals decreased levels of Aβ upon co-culturing compared to old tissue cultured alone. The values are normalized to the levels of Aβ in the old tissue cultured alone and represent mean ± SEM from 2 independent experiments, each experiment was performed in triplicates (**P < 0.01).


Figure 3 ( A , B ) Immunofluorescence analysis of the old APPPS1 slice in co - culture ( 14 DIV ) using antibodies 6E10 ( green ) and CD68 ( red ) . Nuclei were counterstained using DAPI ( blue ) . Image of boxed region in A is depicted at higher magnification in B and reveals clustering of several CD68 positive cells around amyloid plaques . Scale bars : 100 µm ( A ) and 10 µm ( B ) . ( C ) Quantitative analysis of plaque - associated CD68 positive cells in co - culture samples at 7 and 14 DIV reveals a trend towards increased number of CD68 positive cells . The values represent mean ± SEM from at least 3 independent experiments , including total of at least 6 independent slice culture dishes ( n . s . = not significant ) . ( D ) Quantitative analysis of co - culture samples at 7 and 14 DIV reveals a decrease in plaque size . The values represent mean ± SEM from at least 3 independent experiments , including total of at least 6 replicates ( * * * P < 0 . 001 ) . ( E ) Quantitative analysis of plaques surrounded by CD68 positive cells ( clustered plaques ) in co - culture samples at 7 and 14 DIV reveals an increase in the number of clustered plaques . The values are expressed as percentages of clustered plaques from the total number of amyloid plaques . The values represent mean ± SEM from at least 3 independent experiments , including total of at least 6 independent slice culture dishes ( * * * P < 0 . 001 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, B) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) using antibodies 6E10 (green) and CD68 (red). Nuclei were counterstained using DAPI (blue). Image of boxed region in A is depicted at higher magnification in B and reveals clustering of several CD68 positive cells around amyloid plaques. Scale bars: 100 µm (A) and 10 µm (B).(C) Quantitative analysis of plaque-associated CD68 positive cells in co-culture samples at 7 and 14 DIV reveals a trend towards increased number of CD68 positive cells. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 independent slice culture dishes (n.s.= not significant).(D) Quantitative analysis of co-culture samples at 7 and 14 DIV reveals a decrease in plaque size. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 replicates (***P < 0.001).(E) Quantitative analysis of plaques surrounded by CD68 positive cells (clustered plaques) in co-culture samples at 7 and 14 DIV reveals an increase in the number of clustered plaques. The values are expressed as percentages of clustered plaques from the total number of amyloid plaques. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 independent slice culture dishes (***P < 0.001).(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).


Figure 4Either 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Either 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).


Figure 5 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) 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.(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).


Figure 6 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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).(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).


Figure 7 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.


Figure 8 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(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).(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).


Figure 9 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9(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).(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).


Figure 1A . Survival curves for WT and Spns2 - / - CLP models without antibiotic therapy within 72 hours ( n = 13 for each group ) . Data information : Data are presented as percentage ( A N represents biological replicates . P values were determined by log - rank test ( A * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . B . CFU counting in livers and spleens of CLP models without antibiotic therapy ( n = 8 per time point for each group ) . Data information : Data are presented as mean ± s . e . m . ( B , N represents biological replicates . P values were determined by unpaired t - test ( B , * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . C . Concentrations of serum cytokines in CLP models without antibiotic therapy ( n = 6 per time point for each group ) . Data information : Data are presented as mean ± s . e . m . C , N represents biological replicates . P values were determined by unpaired t - test C , * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . E . Survival curves for WT ( n = 12 ) and Spns2 - / - ( n = 17 ) CLP models receiving meropenem treatment within 7 days . Data information : Data are presented as percentage E ) N represents biological replicates . P values were determined by log - rank test E ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . G . Concentrations of serum cytokines in meropenem - treated CLP models ( n = 6 per time point for each group ) . Data information : Data are presented as mean ± s . e . m . G ) . N represents biological replicates . P values were determined by unpaired t - test G ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant .

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

Figure 1A. Survival curves for WT and Spns2-/- CLP models without antibiotic therapy within 72 hours (n=13 for each group). Data information: Data are presented as percentage (A N represents biological replicates. P values were determined by log-rank test (A *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.B. CFU counting in livers and spleens of CLP models without antibiotic therapy (n=8 per time point for each group). Data information: Data are presented as mean ± s.e.m. (B, N represents biological replicates. P values were determined by unpaired t-test (B, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.C. Concentrations of serum cytokines in CLP models without antibiotic therapy (n=6 per time point for each group). Data information: Data are presented as mean ± s.e.m. C, N represents biological replicates. P values were determined by unpaired t-test C, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.E. Survival curves for WT (n=12) and Spns2-/- (n=17) CLP models receiving meropenem treatment within 7 days. Data information: Data are presented as percentage E) N represents biological replicates. P values were determined by log-rank test E) *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.G. Concentrations of serum cytokines in meropenem-treated CLP models (n=6 per time point for each group). Data information: Data are presented as mean ± s.e.m. G). N represents biological replicates. P values were determined by unpaired t-test G). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.


Figure 2A . Gene expression of inflammatory cytokines in isolated PMs challenged with 10 ng / ml LPS ( n = 3 per time point for each group ) . Data information : Data are presented as mean ± s . e . m . ( A , N represents biological replicates . P values were determined by unpaired t - test ( A , * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . B . Survival curves for bone marrow transplanted CLP models without antibiotic therapy within 7 days ( n = 8 for each group ) . Data information : Data are presented as percentage ( B , N represents biological replicates . P values were determined by log - rank test ( B , * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . C . Concentrations of serum cytokines in bone marrow transplanted CLP models without antibiotic therapy ( n = 6 to 8 per time point for each group ) . Data information : Data are presented as mean ± s . e . m . C , N represents biological replicates . P values were determined by unpaired t - test C , * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . D . Survival curves for bone marrow transplanted CLP models receiving meropenem treatment within 7 days ( n = 8 for each group ) . Data information : Data are presented as percentage D , N represents biological replicates . P values were determined by log - rank test D , * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . G . Survival curves for bone marrow transplanted sepsis models induced by E . coli peritoneal infection within 7 days ( n = 4 for each group ) . Data information : Data are presented as percentage G ) N represents biological replicates . P values were determined by log - rank test G ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant .

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

Figure 2A. Gene expression of inflammatory cytokines in isolated PMs challenged with 10 ng/ml LPS (n=3 per time point for each group). Data information: Data are presented as mean ± s.e.m. (A, N represents biological replicates. P values were determined by unpaired t-test (A, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.B. Survival curves for bone marrow transplanted CLP models without antibiotic therapy within 7 days (n=8 for each group). Data information: Data are presented as percentage (B, N represents biological replicates. P values were determined by log-rank test (B, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.C. Concentrations of serum cytokines in bone marrow transplanted CLP models without antibiotic therapy (n=6 to 8 per time point for each group). Data information: Data are presented as mean ± s.e.m. C, N represents biological replicates. P values were determined by unpaired t-test C, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.D. Survival curves for bone marrow transplanted CLP models receiving meropenem treatment within 7 days (n=8 for each group). Data information: Data are presented as percentage D, N represents biological replicates. P values were determined by log-rank test D, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.G. Survival curves for bone marrow transplanted sepsis models induced by E. coli peritoneal infection within 7 days (n=4 for each group). Data information: Data are presented as percentage G) N represents biological replicates. P values were determined by log-rank test G) *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.


Figure 3B . Oxygen consumption rates ( OCR ) and quantifications of basal respiration , maximal respiration , ATP production , and proton leakage in resting WT , Spns2 - / - , and 1 μM S1P pre - treated Spns2 - / - PMs ( n = 4 for each group ) . Data information : Data are presented as mean ± s . e . m . ( B N represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . C . Total glucose consumption in 72 hours ( n = 4 to 5 for each group ) , the ratio of NAD + to NADH ( n = 4 for each group ) , and the levels of intracellular lactate ( n = 4 to 5 for each group ) in resting PMs . Data information : Data are presented as mean ± s . e . m . C ) . N represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant .

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

Figure 3B. Oxygen consumption rates (OCR) and quantifications of basal respiration, maximal respiration, ATP production, and proton leakage in resting WT, Spns2-/-, and 1 μM S1P pre-treated Spns2-/- PMs (n=4 for each group). Data information: Data are presented as mean ± s.e.m. (B N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.C. Total glucose consumption in 72 hours (n=4 to 5 for each group), the ratio of NAD+ to NADH (n=4 for each group), and the levels of intracellular lactate (n=4 to 5 for each group) in resting PMs. Data information: Data are presented as mean ± s.e.m. C). N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.


Figure 4A . Fragmented mitochondrial morphology in Spns2 - / - PMs revealed by ransmission electron microscopy . Zoom - in view of the area in the red frame are shown below . Scale bars : 1 μm . B . The lengths of 38 and 47 mitochondria from WT and Spns2 - / - PMs , respectively ( n = 1 for each group ) . Data information : Data are presented as mean ± s . e . m . N represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . F . The expression of proteins involved in mitochondrial dynamics ( n = 3 for each group ) . Data information : Data are presented as mean ± s . e . m . N represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant . G . Heat map displaying the gene expression of MAS and G3PS components ( n = 3 for each group ) . H . Protein levels of Slc25a12 and Slc25a13 in WT , Spns2 - / - , and 1 μM S1P pre - treated Spns2 - / - PMs ( n = 3 for each group ) . Data information : Data are presented as mean ± s . e . m . N represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . , not significant .

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

Figure 4A. Fragmented mitochondrial morphology in Spns2-/- PMs revealed by ransmission electron microscopy. Zoom-in view of the area in the red frame are shown below. Scale bars: 1 μm. B. The lengths of 38 and 47 mitochondria from WT and Spns2-/- PMs, respectively (n=1 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.F. The expression of proteins involved in mitochondrial dynamics (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.G. Heat map displaying the gene expression of MAS and G3PS components (n=3 for each group).H. Protein levels of Slc25a12 and Slc25a13 in WT, Spns2-/-, and 1 μM S1P pre-treated Spns2-/- PMs (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.


Figure 5D . Activities of total superoxide dismutase ( SOD ) and catalase in resting PMs ( n = 4 for each group ) . Data information : Data are presented as mean ± s . e . m . N represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * * P < 0 . 001 ; n . s . , not significant . E . Protein levels of peroxiredoxin 1 ( Prdx1 ) and Prdx6 in resting PMs ( n = 3 per group ) . Data information : Data are presented as mean ± s . e . m . represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * * P < 0 . 001 ; n . s . , not significant .

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

Figure 5D. Activities of total superoxide dismutase (SOD) and catalase in resting PMs (n=4 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; ***P < 0.001; n.s., not significant.E. Protein levels of peroxiredoxin 1 (Prdx1) and Prdx6 in resting PMs (n=3 per group). Data information: Data are presented as mean ± s.e.m. represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; ***P < 0.001; n.s., not significant.


Figure 6A . Elevated levels of intracellular lactate in resting WT PMs treated with 5 and 10 nM rotenone for 16 hours ( n = 3 for each group ) . Data information : Data are presented as mean ± s . e . m . N represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . C . Enhanced activities of total superoxide dismutase ( SOD ) and catalase in rotenone - treated WT PMs ( n = 3 for each group ) . Data information : Data are presented as mean ± s . e . m . N represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . F . Attenuated activities of total SOD and catalase in oxamate - and S1P - treated Spns2 - / - PMs ( n = 3 to 4 for each group ) . Data information : Data are presented as mean ± s . e . m . N represents biological replicates . P values were determined by unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

Figure 6A. Elevated levels of intracellular lactate in resting WT PMs treated with 5 and 10 nM rotenone for 16 hours (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.C. Enhanced activities of total superoxide dismutase (SOD) and catalase in rotenone-treated WT PMs (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.F. Attenuated activities of total SOD and catalase in oxamate- and S1P-treated Spns2-/- PMs (n=3 to 4 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.


Figure 7A , B . Reduced gene expression of inflammatory cytokines in Spns2 - / - PMs treated with either oxamate ( A ) or mitoquinone ( B ) ( n = 3 per time point for each group ) . Data information : Data are presented as mean ± s . e . m . N represents biological replicates . P values were determined by unpaired t - test . * * P < 0 . 01 ; * * * P < 0 . 001 . C . Activation of Spns2 / S1P signaling and suppression of the lactate - ROS axis promoted the survival of Spns2 - / - sepsis models induced by heat - killed E . coli through the alleviation of hyerinflammation ( n = 5 for Spns2 - / - control group ; n = 4 for each treatment group ) . Data information : Data are presented as mean ± s . e . m . N represents biological replicates . P values were determined by unpaired t - test . * * P < 0 . 01 ; * * * P < 0 . 001 .

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

Figure 7A, B. Reduced gene expression of inflammatory cytokines in Spns2-/- PMs treated with either oxamate (A) or mitoquinone (B) (n=3 per time point for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. **P < 0.01; ***P < 0.001.C. Activation of Spns2/S1P signaling and suppression of the lactate-ROS axis promoted the survival of Spns2-/- sepsis models induced by heat-killed E. coli through the alleviation of hyerinflammation (n=5 for Spns2-/- control group; n=4 for each treatment group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. **P < 0.01; ***P < 0.001.


Figure 8A . S1P treatment restored inflammatory response in Spns2 - / - PMs after 6 hours post - LPS challenge ( n = 3 per time point for each group ) . Data information : Data are presented as mean ± s . e . m . ( A N represents biological replicates . P values were determined by unpaired t - test ( A * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . B . Enhancement of Spns2 / S1P signaling significantly improved the survival of CLP models without antibiotic therapy ( n = 8 for Spns2 - / - saline group ; n = 14 for Spns2 + / - group ; n = 12 for Spns2 - / - S1P treatment group ) . Data information : Data are presented as percentage ( B ) . N represents biological replicates . P values were determined by log - rank test ( B ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . C . Enhancement of Spns2 / S1P signaling restored inflammatory response in Spns2 - / - CLP models at 12 hours post - infection ( n = 6 for each group ) . Data information : Data are presented as mean ± s . e . m . C ) N represents biological replicates . P values were determined by unpaired t - test C ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

Figure 8A. S1P treatment restored inflammatory response in Spns2-/- PMs after 6 hours post- LPS challenge (n=3 per time point for each group). Data information: Data are presented as mean ± s.e.m. (A N represents biological replicates. P values were determined by unpaired t-test (A *P < 0.05; **P < 0.01; ***P < 0.001.B. Enhancement of Spns2/S1P signaling significantly improved the survival of CLP models without antibiotic therapy (n=8 for Spns2-/- saline group; n=14 for Spns2+/- group; n=12 for Spns2-/- S1P treatment group). Data information: Data are presented as percentage (B). N represents biological replicates. P values were determined by log-rank test (B). *P < 0.05; **P < 0.01; ***P < 0.001.C. Enhancement of Spns2/S1P signaling restored inflammatory response in Spns2-/- CLP models at 12 hours post-infection (n=6 for each group). Data information: Data are presented as mean ± s.e.m. C) N represents biological replicates. P values were determined by unpaired t-test C) *P < 0.05; **P < 0.01; ***P < 0.001.


Figure 1 A mRNA expression in FACS - isolated Lin ( Ter119 / CD31 / CD45 ) - Sca1 + progenitor cells from female mouse subcutaneous fat , differentiated in the presence of 1 µM cPGI2 or vehicle for the indicated time , as determined by expression profiling ( n = 3 , E - MTAB - 3693 ) * * * * P = 3 * 10 - 6 ( Day 2 ) , * * * * P = 4 * 10 - 7 ( Day 4 ) , * * * * P = 1 * 10 - 6 ( Day 6 ) in 2x2 ANOVA with Bonferroni posttests ( cPGI2 vs . vehicle ) B mRNA expression in MACS - isolated Lin - Sca1 + progenitor cells from female mouse subcutaneous fat , differentiated in the presence of 100 nM Rosi or vehicle for the indicated time , as determined by qRT - PCR ( n = 4 ) * * * * P = 1 * 10 - 10 ( Day 1 and 2 ) , * * P = 0 . 001 , in 2x2 ANOVA with Bonferroni posttests ( Rosi vs . vehicle ) C mRNA expression in female Lin - Sca1 + cells , differentiated in the presence of 100 nM Rosi or vehicle for 8 days , as determined by qRT - PCR ( n = 3 ) t - test Cited4 - / - vs . Cited4 + / + ( Rosi ) * P = 0 . 013 ( Ucp1 ) , * * P = 0 . 004 ( Cpt1b ) , * P = 0 . 026 ( Dio2 ) D - F mRNA expression in primary SVF cells from human subcutaneous fat , differentiated in the presence of 100 nM Rosi ( D ) or vehicle ( D - F ) , as determined by qRT - PCR at the indicated time points ( n = 5 patients ) . ♀ / ♂ represent individual data ( D ) * * * * P = 3 * 10 - 5 ( Day 2 ) , * * * * P = 3 * 10 - 6 ( Day 6 ) , * * * * P = 4 * 10 - 9 ( Day 10 ) , * * * * P = 3 * 10 - 7 ( Day 14 ) , in 2x2 ANOVA with Bonferroni posttests ( Rosi vs . vehicle ) . ( E , F ) Pearson correlation coefficient ( r ) and P - value are shown G mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days , as determined by qRT - PCR ( n = 3 ) * * * P = 0 . 0002 ( CITED4 ) , * * P = 0 . 002 ( UCP1 ) , * P = 0 . 02 ( UCP1 ) , * P = 0 . 035 / 0 . 026 ( PPARG ) , * * * P = 0 . 0006 ( SLC2A4 ) , * * P = 0 . 002 ( ADIPOQ ) in one - way ANOVA with Tukey posttests ( vs . siCtrl )

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

Figure 1 A mRNA expression in FACS-isolated Lin(Ter119/CD31/CD45)-Sca1+ progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 1 µM cPGI2 or vehicle for the indicated time, as determined by expression profiling (n=3, E-MTAB-3693) ****P=3*10-6 (Day 2), ****P=4*10-7 (Day 4), ****P=1*10-6 (Day 6) in 2x2 ANOVA with Bonferroni posttests (cPGI2 vs. vehicle) B mRNA expression in MACS-isolated Lin-Sca1+ progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT-PCR (n=4) ****P=1*10-10 (Day 1 and 2), **P=0.001, in 2x2 ANOVA with Bonferroni posttests (Rosi vs. vehicle) C mRNA expression in female Lin-Sca1+ cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT-PCR (n=3) t-test Cited4-/- vs. Cited4+/+ (Rosi) *P=0.013 (Ucp1), **P=0.004 (Cpt1b), *P=0.026 (Dio2) D-F mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi (D) or vehicle (D-F), as determined by qRT-PCR at the indicated time points (n=5 patients). ♀/♂ represent individual data (D) ****P=3*10-5 (Day 2), ****P=3*10-6 (Day 6), ****P=4*10-9 (Day 10), ****P=3*10-7 (Day 14), in 2x2 ANOVA with Bonferroni posttests (Rosi vs. vehicle). (E, F) Pearson correlation coefficient (r) and P-value are shown G mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT-PCR (n=3) ***P=0.0002 (CITED4), **P=0.002 (UCP1), *P=0.02 (UCP1), *P=0.035/0.026 (PPARG), ***P=0.0006 (SLC2A4), **P=0.002 (ADIPOQ) in one-way ANOVA with Tukey posttests (vs. siCtrl)


Figure 2 A - C Quantitative fluorescence microscopy of LipidTOX - and DAPI - stained female Cited4F / F Lin - Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days ( n = 5 ) . * * P = 0 . 002 in t - test ( Cre vs . Ctrl ) . Scale bar is 10 µm D , E Ucp1 expression in female Cited4F / F Lin - Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days , as determined by Western blot with VCP as loading control ( n = 3 ) F Cellular respiration in female Lin - Sca1 + progenitor cells differentiated in the presence of 100 nM Rosi for 8 days . The extracellular oxygen consumption rate ( OCR ) was determined upon injection of the indicated substances and normalized to DNA content . CL : CL - 316243

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

Figure 2 A-C Quantitative fluorescence microscopy of LipidTOX- and DAPI-stained female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days (n=5). **P=0.002 in t-test (Cre vs. Ctrl). Scale bar is 10 µm  D, E Ucp1 expression in female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days, as determined by Western blot with VCP as loading control (n=3)  F Cellular respiration in female Lin-Sca1+ progenitor cells differentiated in the presence of 100 nM Rosi for 8 days. The extracellular oxygen consumption rate (OCR) was determined upon injection of the indicated substances and normalized to DNA content. CL: CL-316243


Figure 4 A , B mRNA expression in scWAT of mice fed a diet with 0 . 0075 % Rosi or control diet for 2 . 5 weeks , determined by qRT - PCR t - test Cited4 - / - vs . Cited4 + / + ( Rosi ) , ( A ) n = 5 / 5 / 6 / 6 , * * P = 0 . 003 ( Ucp1 ) , * * P = 0 . 008 ( Cidea ) , * * P = 0 . 007 ( Cyc1 ) , * P = 0 . 048 ( Ndufb3 ) , ( B ) n = 5 / 4 / 5 / 5 , * P = 0 . 022 ( Ucp1 ) , * P = 0 . 037 ( Cidea ) C , D Ucp1 expression in scWAT of mice treated as in A , as determined by Western blot with VCP as loading control t - test Cited4 - / - vs . Cited4 + / + ( Rosi ) , females : n = 5 / 5 / 6 / 6 , * P = 0 . 011 , males : n = 5 / 4 / 5 / 5 E mRNA expression in scWAT of female mice treated with CL - 316 , 243 ( CL ) ( 1 mg / kg / day via Alzet minipumps ) or vehicle for 10 days ( n = 5 / 7 / 13 , t - test Cited4 - / - vs . Cited4 + / + ( CL ) ) F mRNA expression in scWAT of female mice exposed to 5 ° C or 23 ° C for 2 weeks ( n = 5 / 10 / 6 , t - test Cited4 - / - vs . Cited4 + / + ( 5 ° C ) )

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

Figure 4 A, B mRNA expression in scWAT of mice fed a diet with 0.0075% Rosi or control diet for 2.5 weeks, determined by qRT-PCR t-test Cited4-/- vs. Cited4+/+ (Rosi), (A) n=5/5/6/6, **P=0.003 (Ucp1), **P=0.008 (Cidea), **P=0.007 (Cyc1), *P=0.048 (Ndufb3), (B) n=5/4/5/5, *P=0.022 (Ucp1), *P=0.037 (Cidea) C, D Ucp1 expression in scWAT of mice treated as in A, as determined by Western blot with VCP as loading control t-test Cited4-/- vs. Cited4+/+ (Rosi), females: n=5/5/6/6, *P=0.011, males: n=5/4/5/5 E mRNA expression in scWAT of female mice treated with CL-316,243 (CL) (1 mg/kg/day via Alzet minipumps) or vehicle for 10 days (n=5/7/13, t-test Cited4-/- vs. Cited4+/+ (CL))  F mRNA expression in scWAT of female mice exposed to 5°C or 23°C for 2 weeks (n=5/10/6, t-test Cited4-/- vs. Cited4+/+ (5°C))


Figure 5 A Oxygen consumption rate of female mice fed a diet with 0 . 0075 % Rosi for 2 . 5 weeks , determined by indirect calorimetry and normalized to body weight ( n = 9 / 10 ) . Grey bars represent the dark phase B Oxygen consumption rate of female mice shown in ( A ) adjusted for body weight by ANCOVA . 3 - day averages of VO2 were calculated for each mouse ( n = 9 / 10 ) . * P = 0 . 025 in ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + ) C Oxygen consumption rate of female mice fed a diet with 0 . 0075 % Rosi for 3 . 5 weeks and injected with 1 mg / kg CL - 316 , 243 ( CL ) at the indicated time point , as determined by indirect calorimetry ( n = 9 / 10 ) . * P = 0 . 013 ( - 1 h ) , * P = 0 . 045 ( 3 h ) , * P = 0 . 015 ( 5 h ) , * P = 0 . 016 ( 6 h ) , * P = 0 . 019 ( 8 h ) , * P = 0 . 046 ( 10 h ) in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) D Maximal CL - induced oxygen consumption rate of female mice shown in ( C ) adjusted for body weight by ANCOVA . Averages of VO2 throughout 3 - 8 hours post CL injection were calculated for each mouse ( n = 9 / 10 ) . * P = 0 . 049 in ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + ) E Oxygen consumption rate of male mice treated as in ( A ) , determined as in ( C ) ( n = 9 / 10 ) . ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + ) F Oxygen consumption rate of male mice treated as in ( C ) , determined as in ( C ) ( n = 9 / 10 ) . * P = 0 . 044 ( 9 h ) , * P = 0 . 013 ( 12 h ) in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) G Maximal CL - induced oxygen consumption rate of male mice shown in ( F ) and calculated as in ( D ) ( n = 9 / 10 ) . ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + )

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

Figure 5 A Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 2.5 weeks, determined by indirect calorimetry and normalized to body weight (n=9/10). Grey bars represent the dark phase  B Oxygen consumption rate of female mice shown in (A) adjusted for body weight by ANCOVA. 3-day averages of VO2 were calculated for each mouse (n=9/10). *P=0.025 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)  C Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 3.5 weeks and injected with 1 mg/kg CL-316,243 (CL) at the indicated time point, as determined by indirect calorimetry (n=9/10). *P=0.013 (-1 h), *P=0.045 (3 h), *P=0.015 (5 h), *P=0.016 (6 h), *P=0.019 (8 h), *P=0.046 (10 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)  D Maximal CL-induced oxygen consumption rate of female mice shown in (C) adjusted for body weight by ANCOVA. Averages of VO2 throughout 3-8 hours post CL injection were calculated for each mouse (n=9/10). *P=0.049 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)  E Oxygen consumption rate of male mice treated as in (A), determined as in (C) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)  F Oxygen consumption rate of male mice treated as in (C), determined as in (C) (n=9/10). *P=0.044 (9 h), *P=0.013 (12 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)  G Maximal CL-induced oxygen consumption rate of male mice shown in (F) and calculated as in (D) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)


Figure 6 A , B mRNA expression in scWAT of mice fed a high - fat diet ( HFD ) for 11 weeks followed by 5 weeks of HFD with 0 . 0075 % Rosi ( qRT - PCR ) . t - test Cited4 - / - vs . Cited4 + / + , ( A ) n = 8 / 7 , * P = 0 . 029 ( Ucp1 ) , * * P = 0 . 009 ( Elovl3 ) , * * P = 0 . 006 ( Cpt1b ) , * * P = 0 . 004 ( Cyc1 ) , * P = 0 . 016 ( Ndufb3 ) , * * P = 0 . 003 ( Cd36 ) , * * P = 0 . 009 ( Gyk ) , ( B ) n = 7 / 8 , * P = 0 . 026 ( Pck1 ) C , D Ucp1 protein expression in scWAT of mice treated as in ( A ) , determined by Western blot with VCP as loading control ( n = 8 / 7 for females and n = 7 / 8 for males ) . Arrow indicates the band specific to Ucp1 ( see Appendix Fig 4F )

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

Figure 6 A, B mRNA expression in scWAT of mice fed a high-fat diet (HFD) for 11 weeks followed by 5 weeks of HFD with 0.0075% Rosi (qRT-PCR). t-test Cited4-/- vs. Cited4+/+, (A) n=8/7, *P=0.029 (Ucp1), **P=0.009 (Elovl3), **P=0.006 (Cpt1b), **P=0.004 (Cyc1), *P=0.016 (Ndufb3), **P=0.003 (Cd36), **P=0.009 (Gyk), (B) n=7/8, *P=0.026 (Pck1)  C, D Ucp1 protein expression in scWAT of mice treated as in (A), determined by Western blot with VCP as loading control (n=8/7 for females and n=7/8 for males). Arrow indicates the band specific to Ucp1 (see Appendix Fig 4F)


Figure 7 A Blood glucose during glucose tolerance test ( GTT ) on female mice after 11 weeks of HFD and 3 weeks of HFD + Rosi ( n = 8 ) . * * * P = 0 . 001 , in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) B Fasting serum insulin in female mice after 10 weeks of HFD ( " Before Rosi " ) or 11 weeks of HFD and 2 . 5 weeks of HFD + Rosi ( " After Rosi " ) ( n = 8 ) . * * * P = 0 . 0009 in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi ) ; P = 0 . 725 ( Cited4 - / - vs . Cited4 + / + Before Rosi ) C Blood glucose during GTT on male mice as in ( A ) ( n = 5 / 8 ) . Repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) D Fasting serum insulin in male mice as in ( C ) ( n = 7 / 7 / 8 / 8 ) . * * * P = 0 . 001 ( Cited4 + / + ) , * * * P = 0 . 0008 ( Cited4 - / - ) in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi ) E , F Blood glucose during insulin tolerance test ( ITT ) with 1 U insulin per kg body mass on female mice after 12 weeks of HFD ( n = 8 ) ( E ) or 16 weeks of HFD and 4 weeks of HFD + Rosi ( n = 7 ) ( F ) , expressed as % of the 0 - time point value . * * P = 0 . 003 in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) . G Area under the curve ( AUC ) of blood glucose during the ITT ( 0 - 120 min ) in ( E , F ) . 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi ) H , I Blood glucose during insulin tolerance test ( ITT ) on male mice as in ( E , F ) , n = 8 ( H ) , n = 7 / 8 ( I ) . Repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) . J Area under the curve ( AUC ) of blood glucose during the ITT in ( H , J ) . * * P = 0 . 001 , * * * P = 0 . 00006 in 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi ) K , L Fasting serum non - esterified fatty acids ( NEFA ) in female ( K ) or male ( L ) mice after 10 weeks of HFD ( " Before Rosi " ) or 11 weeks of HFD and 2 . 5 weeks of HFD + Rosi ( " After Rosi " ) , n = 7 / 8 / 8 / 8 ( K ) , n = 6 / 6 / 6 / 8 ( L ) . * * P = 0 . 001 in 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi )

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

Figure 7 A Blood glucose during glucose tolerance test (GTT) on female mice after 11 weeks of HFD and 3 weeks of HFD+Rosi (n=8). ***P=0.001, in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)  B Fasting serum insulin in female mice after 10 weeks of HFD ("Before Rosi") or 11 weeks of HFD and 2.5 weeks of HFD+Rosi ("After Rosi") (n=8). ***P=0.0009 in repeated measures 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi); P=0.725 (Cited4-/- vs. Cited4+/+ Before Rosi)  C Blood glucose during GTT on male mice as in (A) (n=5/8). Repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)  D Fasting serum insulin in male mice as in (C) (n=7/7/8/8). ***P=0.001 (Cited4+/+), ***P=0.0008 (Cited4-/-) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)  E, F Blood glucose during insulin tolerance test (ITT) with 1 U insulin per kg body mass on female mice after 12 weeks of HFD (n=8) (E) or 16 weeks of HFD and 4 weeks of HFD+Rosi (n=7) (F), expressed as % of the 0-time point value. **P=0.003 in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). G Area under the curve (AUC) of blood glucose during the ITT (0-120 min) in (E,F). 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)  H, I Blood glucose during  insulin tolerance test (ITT) on male mice as in (E,F), n=8 (H), n=7/8 (I). Repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). J Area under the curve (AUC) of blood glucose during the ITT in (H,J). **P=0.001, ***P=0.00006 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)  K, L Fasting serum non-esterified fatty acids (NEFA) in female (K) or male (L) mice after 10 weeks of HFD ("Before Rosi") or 11 weeks of HFD and 2.5 weeks of HFD+Rosi ("After Rosi"), n=7/8/8/8 (K), n=6/6/6/8 (L). **P=0.001 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)


f1 ( a ) Δ133p53α expression is induced by cycloheximide ( CHX ) . MRC - 5 fibroblasts ( at population doubling levels ( PDLs ) 51 ) were treated with CHX ( 25 μg ml − 1 ) for indicated time periods and examined in immunoblot for Δ133p53α and full - length p53 . β - actin was a loading control for normalization . The relative expression levels of Δ133p53α and full - length p53 are shown below the images . ( b ) Δ133p53α expression is induced by phosphoinositide 3 - kinase inhibitors . MRC - 5 fibroblasts ( PDLs 51 ) were treated for 4 h with LY303511 ( 10 μM ) , LY294002 ( 10 μM ) , Wortmannin ( 50 nM ) or dimethylsulphoxide alone ( as control ) and examined in immunoblot for Δ133p53α and β - actin expression . ( c ) Diminished Δ133p53α at replicative senescence is restored by treatment with bafilomycin A1 . The immunoblot analyses were performed in early - passage ( Y ) and replicatively senescent ( S ) human fibroblast strains MRC - 5 and WI - 38 . The examined PDLs were 30 ( Y ) and 60 ( S ) for MRC - 5 ; and 30 ( Y ) and 54 ( S ) for WI - 38 . Bafilomycin A 1 treatment was at 100 nM for 4 h . The relative expression levels of Δ133p53α , full - length p53 and p62 / SQSTM1 ( normalized with β - actin ) are shown . Anti - LC3B antibody detected LC3 - I and LC3 - II , which was used in quantitative analysis shown in d . ( d ) Replicative senescence is not associated with enhanced autophagy in general . LC3 - II expression levels were normalized with β - actin ( data in c ) and are shown in the bar graphs as relative values to replicatively senescent cells without bafilomycin A1 ( S / − ; defined as 1 ) . The LC3 - II levels in replicatively senescent cells were higher than those in early - passage cells whether in the presence or absence of bafilomycin A1 , which may suggest increased synthesis of autophagosomes at replicative senescence . However , the differences in LC3 - II levels between the presence and absence of bafilomycin A1 were similar in early - passage and replicatively senescent cells : in MRC - 5 , 1 . 9 for Y ( 2 . 1 − 0 . 2 ) and 2 . 0 for S ( 3 . 0 − 1 ) ; in WI - 38 , 1 . 6 for Y ( 2 . 2 − 0 . 6 ) and 1 . 6 for S ( 2 . 6 − 1 ) . These data indicate that early - passage and replicatively senescent cells have similar rates of autophagic flux , that is , the number of autophagosomes that are delivered to lysosomes for degradation .

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

f1(a) Δ133p53α expression is induced by cycloheximide (CHX). MRC-5 fibroblasts (at population doubling levels (PDLs) 51) were treated with CHX (25 μg ml−1) for indicated time periods and examined in immunoblot for Δ133p53α and full-length p53. β-actin was a loading control for normalization. The relative expression levels of Δ133p53α and full-length p53 are shown below the images.(b) Δ133p53α expression is induced by phosphoinositide 3-kinase inhibitors. MRC-5 fibroblasts (PDLs 51) were treated for 4 h with LY303511 (10 μM), LY294002 (10 μM), Wortmannin (50 nM) or dimethylsulphoxide alone (as control) and examined in immunoblot for Δ133p53α and β-actin expression.(c) Diminished Δ133p53α at replicative senescence is restored by treatment with bafilomycin A1. The immunoblot analyses were performed in early-passage (Y) and replicatively senescent (S) human fibroblast strains MRC-5 and WI-38. The examined PDLs were 30 (Y) and 60 (S) for MRC-5; and 30 (Y) and 54 (S) for WI-38. Bafilomycin A 1 treatment was at 100 nM for 4 h. The relative expression levels of Δ133p53α, full-length p53 and p62/SQSTM1 (normalized with β-actin) are shown. Anti-LC3B antibody detected LC3-I and LC3-II, which was used in quantitative analysis shown in d. (d) Replicative senescence is not associated with enhanced autophagy in general. LC3-II expression levels were normalized with β-actin (data in c) and are shown in the bar graphs as relative values to replicatively senescent cells without bafilomycin A1 (S/−; defined as 1). The LC3-II levels in replicatively senescent cells were higher than those in early-passage cells whether in the presence or absence of bafilomycin A1, which may suggest increased synthesis of autophagosomes at replicative senescence. However, the differences in LC3-II levels between the presence and absence of bafilomycin A1 were similar in early-passage and replicatively senescent cells: in MRC-5, 1.9 for Y (2.1−0.2) and 2.0 for S (3.0−1); in WI-38, 1.6 for Y (2.2−0.6) and 1.6 for S (2.6−1). These data indicate that early-passage and replicatively senescent cells have similar rates of autophagic flux, that is, the number of autophagosomes that are delivered to lysosomes for degradation.


f2 ( a - c ) Knockdown of pro - autophagic proteins restores Δ133p53α expression . MRC - 5 fibroblasts at late - passage ( PDLs 51 ) were transfected with three independent siRNA oligonucleotides ( lanes 1 - 3 ) against ATG5 ( a ) , ATG7 ( b ) and Beclin - 1 ( c ) , along with their control siRNA ( lane C ) , for 4 days . Knockdown of ATG5 was confirmed by the loss of ATG5 - ATG12 conjugate ( detected by anti - ATG5 antibody ) . Knockdown of the other two proteins was confirmed using the corresponding antibodies . Δ133p53α , full - length p53 and LC3B ( LC3 - I and LC3 - II ) were examined in all samples . The relative expression levels of Δ133p53α and full - length p53 ( normalized with β - actin ) are shown below the images . ( d ) Knockdown of pro - autophagic proteins and treatment with bafilomycin A1 show similar effects on Δ133p53α and p62 / SQSTM1 . MRC - 5 fibroblasts at late - passage ( PDLs 51 ) were transfected with control siRNA ( c ) , ATG5 siRNA ( A5 , no . 1 in a ) , ATG7 siRNA ( A7 , no . 1 in b ) and Beclin - 1 siRNA ( B1 , no . 2 in c ) as in a - c , untreated ( − ) or treated with bafilomycin A1 ( + , 100 nM for 4 h ) , and examined in immunoblots for indicated proteins . The relative expression levels of Δ133p53α and p62 / SQSTM1 ( normalized with β - actin ) are shown below the images .

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

f2(a-c) Knockdown of pro-autophagic proteins restores Δ133p53α expression. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with three independent siRNA oligonucleotides (lanes 1-3) against ATG5 (a), ATG7 (b) and Beclin-1 (c), along with their control siRNA (lane C), for 4 days. Knockdown of ATG5 was confirmed by the loss of ATG5-ATG12 conjugate (detected by anti-ATG5 antibody). Knockdown of the other two proteins was confirmed using the corresponding antibodies. Δ133p53α, full-length p53 and LC3B (LC3-I and LC3-II) were examined in all samples. The relative expression levels of Δ133p53α and full-length p53 (normalized with β-actin) are shown below the images.(d) Knockdown of pro-autophagic proteins and treatment with bafilomycin A1 show similar effects on Δ133p53α and p62/SQSTM1. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with control siRNA (c), ATG5 siRNA (A5, no. 1 in a), ATG7 siRNA (A7, no. 1 in b) and Beclin-1 siRNA (B1, no. 2 in c) as in a-c, untreated (−) or treated with bafilomycin A1 (+, 100 nM for 4 h), and examined in immunoblots for indicated proteins. The relative expression levels of Δ133p53α and p62/SQSTM1 (normalized with β-actin) are shown below the images.