1205Lu cells were transfected , or not , with 2 different siRNA targeted against DRP1 ( siDRP1 # 1 or 2 ) or an untargeted siRNA ( siUT ) . Top , 48h after transfection , protein extracts were prepared and DRP1 expression was evaluated by western blot . Bottom , 36h post transfection , cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and cell motility was tracked by video microscopy ( n = 40 cells per group ) .

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1205Lu cells were transfected, or not, with 2 different siRNA targeted against DRP1 (siDRP1#1 or 2) or an untargeted siRNA (siUT). Top, 48h after transfection, protein extracts were prepared and DRP1 expression was evaluated by western blot. Bottom, 36h post transfection, cells were exposed to 3T3-F442A EV (3T3-EV) and cell motility was tracked by video microscopy (n = 40 cells per group).


1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) and immediately treated , or not , with Mdivi - 1 . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) .

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1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) and immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group).


Left panel , indicated melanoma cells exposed to 3T3 - F442A EV were fixed , stained with BODIPY and Phalloidin before observation by confocal microscopy . Right panel , quantification of the percentage of cells presenting lipid droplets ( LD ) within membrane protrusions ( n = 6 ) .

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Left panel, indicated melanoma cells exposed to 3T3-F442A EV were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=6).


Left panel , melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) were fixed , stained with BODIPY and Phalloidin before observation by confocal microscopy . Right panel , quantification of the percentage of cells presenting lipid droplets ( LD ) within membrane protrusions ( n = 5 for SKMEL28 and n = 6 for 1205Lu ) .

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Left panel, melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=5 for SKMEL28 and n=6 for 1205Lu).


1205Lu cells exposed to 3T3 - F442A EV were stained with a MitoTracker probe . Then , cells were fixed and stained with BODIPY before observation by confocal microscopy . A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown ( scale bar : 5µm ) .

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1205Lu cells exposed to 3T3-F442A EV were stained with a MitoTracker probe. Then, cells were fixed and stained with BODIPY before observation by confocal microscopy. A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown (scale bar: 5µm).


Transmission electron microscope observations of 1205Lu cells exposed , or not , to 3T3 - F442A EV . Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right .

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Transmission electron microscope observations of 1205Lu cells exposed, or not, to 3T3-F442A EV. Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right.


Number of lipid droplets ( LD ) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( 3T3 - EV ) ( n = 28 for control and n = 27 for 3T3 - EV ) . Area of lipid droplets ( LD ) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( n = 19 for control and n = 25 for 3T3 - EV ) .

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Number of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV) (n=28 for control and n=27 for 3T3-EV). Area of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (n=19 for control and n=25 for 3T3-EV).


Left panel , 1205Lu melanoma cells were exposed , or not , to 3T3 - F442A EV . Then , live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy . Right panel , quantification of the percentage of cells presenting lysosome within membrane protrusions ( n = 3 ) .

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Left panel, 1205Lu melanoma cells were exposed, or not, to 3T3-F442A EV. Then, live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy. Right panel, quantification of the percentage of cells presenting lysosome within membrane protrusions (n=3).


TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma . Statistical significance of survival was evaluated with a Log - rank ( Mantel - Cox ) Test ( n = 449 , 450 and 459 for HADHA , HADHB and ECHS1 respectively ) .

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TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=449, 450 and 459 for HADHA, HADHB and ECHS1 respectively).


BODIPY staining of neutral lipid stores in the indicated melanoma cell lines , which present increasing degrees of aggressiveness ( from left to right ) . DAPI was used to counterstain nuclei . Scale bar : 20µm .

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BODIPY staining of neutral lipid stores in the indicated melanoma cell lines, which present increasing degrees of aggressiveness (from left to right). DAPI was used to counterstain nuclei. Scale bar: 20µm.


FAO in the indicated melanoma cell lines was measured . Bars and error bars represent means ± SEM ( n = 5 ) ; statistically significant by one - way ANOVA with post hoc Tukey ' s test , * * * P < 0 . 001 . If not indicated , non - significant .

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FAO in the indicated melanoma cell lines was measured. Bars and error bars represent means ± SEM (n=5); statistically significant by one-way ANOVA with post hoc Tukey's test, ***P < 0.001. If not indicated, non-significant.


TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma . Statistical significance of survival was evaluated with a Log - rank ( Mantel - Cox ) Test ( n = 459 ) .

TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=459).


( A ) RT - PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages . NC indicates the no template control .

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(A) RT-PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages. NC indicates the no template control.


( B ) Time - course expression of sinhcaf revealed by whole - mount in situ hybridization . Scale bars , 100

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(B) Time-course expression of sinhcaf revealed by whole-mount in situ hybridization. Scale bars, 100 μ


( C ) RT - PCR analysis for spatial expression of sinhcaf mRNA in adult tissues .

(C) RT-PCR analysis for spatial expression of sinhcaf mRNA in adult tissues.


( E ) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf - / - mutant embryos as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

(E) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf -/- mutant embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test.


( F ) Fluorescence microscopy of 10hpf Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos . Scale bar , 400 μm .

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(F) Fluorescence microscopy of 10hpf Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Scale bar, 400 μm.


( G ) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg ( CMV : EGFP ; CMV : sinhcaf ) embryos as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using 2 - way ANOVA followed by Holm - Sidak post - hoc test .

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(G) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg(CMV:EGFP;CMV:sinhcaf) embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test.


( H ) Percentage of male zebrafish in WT ( n = 274 ) , MZsinhcaf - / - ( n = 166 ) , Tg ( CMV : EGFP ; CMV : sinhcaf ) ( n = 158 ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) ( n = 135 ) adult zebrafish . Data shown are mean ± SEM ( n = the number of fish analyzed ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 .

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(H) Percentage of male zebrafish in WT (n = 274), MZsinhcaf -/- (n = 166), Tg(CMV:EGFP;CMV:sinhcaf) (n = 158) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) (n = 135) adult zebrafish. Data shown are mean ± SEM (n = the number of fish analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.


( A and B ) Whole - mount in situ hybridization was performed in WT , MZsinhcaf - / - , Tg ( CMV : EGFP ; CMV : sinhcaf ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos with PGCs markers nanos3 ( A ) and vasa ( B ) . Scale bars , 100 μm . ( C ) Quantitative analysis of PGCs in bud stage WT , MZsinhcaf - / - , Tg ( CMV : EGFP ; CMV : sinhcaf ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos . Data shown are mean ± SEM ( n = 10 , the number of embryos analyzed ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 .

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(A and B) Whole-mount in situ hybridization was performed in WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos with PGCs markers nanos3 (A) and vasa (B). Scale bars, 100 μm. (C) Quantitative analysis of PGCs in bud stage WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Data shown are mean ± SEM (n = 10, the number of embryos analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.


( D ) Maximal intensity projection of a confocal z - stack of 24hpf Tg ( piwil1 : EGFP ) and MZsinhcaf - / - ; Tg ( piwil1 : EGFP ) zebrafish embryos , dorsal views . Arrowheads point to PGCs . Scale bars , 100 μm . ( E ) Quantitative analysis of PGCs in 24hpf Tg ( piwil1 : EGFP ) and MZsinhcaf - / - ; Tg ( piwil1 : EGFP ) zebrafish embryos . Data shown are mean ± SEM ( n = 13 , the number of embryos analyzed ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

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(D) Maximal intensity projection of a confocal z-stack of 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP) zebrafish embryos, dorsal views. Arrowheads point to PGCs. Scale bars, 100 μm. (E) Quantitative analysis of PGCs in 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP)zebrafish embryos. Data shown are mean ± SEM (n = 13, the number of embryos analyzed). Statistical analysis was performed using unpaired two-tailed Student's t-test.


( A ) Whole - mount in situ hybridization was performed in 4 - cell stage WT , MZsinhcaf - / - zebrafish embryos with germ plasm marker vasa . Red arrowheads point to cleavage furrows in the 4 - cell embryos . Scale bars , 100

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(A) Whole-mount in situ hybridization was performed in 4-cell stage WT, MZsinhcaf -/- zebrafish embryos with germ plasm marker vasa. Red arrowheads point to cleavage furrows in the 4-cell embryos. Scale bars, 100 μm


( B ) Relative mRNA levels of germ plasm genes vasa , dnd , nanos3 , dazl and buc in WT and MZsinhcaf - / - mutant eggs , respectively , as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using 2 - way ANOVA followed by the Holm - Sidak post - hoc test ; NS , no significant difference .

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(B) Relative mRNA levels of germ plasm genes vasa, dnd, nanos3, dazl and buc in WT and MZsinhcaf -/- mutant eggs, respectively, as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by the Holm-Sidak post-hoc test; NS, no significant difference.


( C ) Maximal intensity projection of a confocal z - stack of 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP mRNA . White arrowheads point to cleavage furrows of 4 - cell embryos . Scale bars , 100 μm .

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(C) Maximal intensity projection of a confocal z-stack of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. White arrowheads point to cleavage furrows of 4-cell embryos. Scale bars, 100 μm.


( D ) Relative GFP - Buc density at the cleavage furrow of 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP mRNA . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data shown are mean ± SEM ( n = 12 , the number of embryos analyzed ) .

(D) Relative GFP-Buc density at the cleavage furrow of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. Statistical analysis was performed using unpaired two-tailed Student's t-test. Data shown are mean ± SEM (n = 12, the number of embryos analyzed).


( E ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test .

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(E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test.


( F ) Western blots for GFP - Buc expression in 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP

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(F) Western blots for GFP-Buc expression in 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA


( A , B and C ) Co - immunoprecipitation of zebrafish Sinhcaf - GFP with HA - Hdac1 ( A ) , HA - Sin3a ( B ) or HA - Sap18 ( C ) in HEK293Tcells transfected with both Sinhcaf - GFP and HA - Hdac1 ( A ) , HA - Sin3a ( B ) or HA - Sap18 ( C ) plasmids .

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(A, B and C) Co-immunoprecipitation of zebrafish Sinhcaf-GFP with HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) in HEK293Tcells transfected with both Sinhcaf-GFP and HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) plasmids.


( D ) Relative mRNA level of sinhcaf in zebrafish PG , PV , EV , MV and FG follicles was measured using RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) .

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(D) Relative mRNA level of sinhcaf in zebrafish PG, PV, EV, MV and FG follicles was measured using RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).


( E and F ) Western blots for acetyl - histone H3 ( K9 ) ( Ace - H3 - K9 ) , histone H3 in WT ( E ) or MZsinhcaf - / - ( F ) PG , PV , EV , MV and FG follicles . α - tubulin was used as a loading control . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) .

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(E and F) Western blots for acetyl-histone H3 (K9) (Ace-H3-K9), histone H3 in WT (E) or MZsinhcaf -/- (F) PG, PV, EV, MV and FG follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).


( G and H ) Statistical analysis of the relative levels of Ace - H3 - K9 in WT ( G ) or MZsinhcaf - / - ( H ) PG , PV , EV , MV and FG follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) .

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(G and H) Statistical analysis of the relative levels of Ace-H3-K9 in WT (G) or MZsinhcaf -/- (H) PG, PV, EV, MV and FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).


( I , J and K ) Western blots for Ace - H3 - K9 and H3 in WT and MZsinhcaf - / - EV ( I ) , MV ( J ) and FG ( K ) follicles . α - tubulin was used as a loading control . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III )

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(I, J and K) Western blots for Ace-H3-K9 and H3 in WT and MZsinhcaf -/- EV (I), MV (J) and FG (K) follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III)


( L ) Western blots for Ace - H4 - K5 , Ace - H3 - K18 and H3 in WT and MZsinhcaf - / - FG follicles . α - tubulin was used as a loading control . Data information : FG , full - grown stage ( late stage III )

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(L) Western blots for Ace-H4-K5, Ace-H3-K18 and H3 in WT and MZsinhcaf -/- FG follicles. α-tubulin was used as a loading control. Data information: FG, full-grown stage (late stage III)


( M , N and O ) Statistical analysis of the relative levels of Ace - H3 - K9I n WT and MZsinhcaf - / - EV ( M ) , MV ( N ) and FG ( O ) follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III )

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(M, N and O) Statistical analysis of the relative levels of Ace-H3-K9I n WT and MZsinhcaf -/- EV (M), MV (N) and FG (O) follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III)


( P and Q ) Statistical analysis of the relative levels of Ace - H3 - K18 ( P ) and Ace - H4 - K5 ( Q ) in WT and MZsinhcaf - / - FG follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data information : ; FG , full - grown stage ( late stage III )

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

(P and Q) Statistical analysis of the relative levels of Ace-H3-K18 (P) and Ace-H4-K5 (Q) in WT and MZsinhcaf -/- FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: ; FG, full-grown stage (late stage III)


( A ) Maximal intensity projection of a confocal z - stack of 24hpf Tg ( piwil : egfp ) , Tg ( piwil : egfp ) injected with kif26ab MO and Tg ( piwil : egfp ) injected with control MO embryos . ( B ) Quantitative analysis of PGCs in 24hpf Tg ( piwil : egfp ) ( n = 11 ) , Tg ( piwil : egfp ) injected with kif26ab MO ( n = 12 ) and Tg ( piwil : egfp ) injected with control MO ( n = 13 ) embryos . Data shown are mean ± SEM ( n = the number of embryos analyzed ) .

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

(A) Maximal intensity projection of a confocal z-stack of 24hpf Tg(piwil:egfp), Tg(piwil:egfp) injected with kif26ab MO and Tg(piwil:egfp) injected with control MO embryos. (B) Quantitative analysis of PGCs in 24hpf Tg(piwil:egfp) (n = 11), Tg(piwil:egfp) injected with kif26ab MO (n = 12) and Tg(piwil:egfp) injected with control MO (n = 13) embryos. Data shown are mean ± SEM (n = the number of embryos analyzed).


( C ) Maximal intensity projection of a confocal z - stack of 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . White arrowheads point to cleavage furrows of 4 - cell embryos .

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

(C) Maximal intensity projection of a confocal z-stack of 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. White arrowheads point to cleavage furrows of 4-cell embryos.


( D ) Relative GFP - Buc density at the cleavage furrow of 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA ( n = 11 ) , GFP - Buc mRNA + RFP mRNA + Control MO ( n = 11 ) and GFP - Buc mRNA + RFP mRNA + kif26ab MO ( n = 10 ) . Data shown are mean ± SEM ( n = the number of embryos analyzed ) .

(D) Relative GFP-Buc density at the cleavage furrow of 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA (n = 11), GFP-Buc mRNA + RFP mRNA + Control MO (n = 11) and GFP-Buc mRNA + RFP mRNA + kif26ab MO (n = 10). Data shown are mean ± SEM (n = the number of embryos analyzed).


( E ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . ( F ) Western blots for GFP - Buc expression in 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . Data information : Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . Scale bars , 100 μm .

(E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (F) Western blots for GFP-Buc expression in 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.


( G ) Maximal intensity projection of a confocal z - stack of MZsinhcaf - / - or WT 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . White arrowheads point to cleavage furrows of 4 - cell embryos .

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

(G) Maximal intensity projection of a confocal z-stack of MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. White arrowheads point to cleavage furrows of 4-cell embryos.


( H ) Relative GFP - Buc density at the cleavage furrow of MZsinhcaf - / - ( n = 12 ) or WT ( n = 13 ) 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . Data shown are mean ± SEM ( n = the number of embryos analyzed ) .

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

(H) Relative GFP-Buc density at the cleavage furrow of MZsinhcaf -/- (n = 12) or WT (n = 13) 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. Data shown are mean ± SEM (n = the number of embryos analyzed).


( I ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . ( J ) Western blots for GFP - Buc expression in MZsinhcaf - / - or WT 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . Data information : Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . Scale bars , 100 μm .

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

(I) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (J) Western blots for GFP-Buc expression in MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.


A . Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease ( MNase ) for three types of DNA wrapped around the CENP - A nucleosome ( red ) and the H3 nucleosome ( green ) . For α - satellite DNA with initial size of 171 bp , size ranges ( 141 - 171 ) corresponding to DNA lengths above NCP ( nucleosome core particle ) is presented . Data are presented as mean ( SD ) for each time point based on three independent experiments .

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

A. Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease (MNase) for three types of DNA wrapped around the CENP-A nucleosome (red) and the H3 nucleosome (green). For α-satellite DNA with initial size of 171 bp, size ranges (141-171) corresponding to DNA lengths above NCP (nucleosome core particle) is presented. Data are presented as mean (SD) for each time point based on three independent experiments.


B . Native PAGE gel stained with Commassie blue showing complexes formed between CENP - A or H3 nucleosome and CENP - CCR . Lane 1 : CENP - A nucleosome , Lanes 2 - 4 : Increasing amounts of CENP - CCR are added to CENP - A nucleosome . Generation of a sharp band with slower mobility indicates formation of a specific CENP - A / CENP - CCR complex . Lane 5 : H3 nucleosome . Lanes 6 - 8 : Increasing amounts of CENP - CCR are added to H3 nucleosome . Smear on the gel indicates formation of non - specific CENP - A / CENP - CCR complexes . C . Same experiment as in ( B ) using CENP - Cmotif . Lane 1 . CENP - A nucleosome . Lanes 2 - 4 . Increasing amounts of CENP - Cmotif are added to CENP - A nucleosome . Upon binding CENP - Cmotif , CENP - A nucleosome migrates slower through the gel . Note only modest change in mobility due to small size of CENP - Cmotif , comparing to CENP - CCR in ( B ) . Lane 5 . H3 nucleosome . Lanes 6 - 8 . Increasing amounts of CENP - Cmotif are added to H3 nucleosome . Smear on the gel indicates formation of non - specific CENP - A / CENP - Cmotif complexes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Native PAGE gel stained with Commassie blue showing complexes formed between CENP-A or H3 nucleosome and CENP-CCR. Lane 1: CENP-A nucleosome, Lanes 2-4: Increasing amounts of CENP-CCR are added to CENP-A nucleosome. Generation of a sharp band with slower mobility indicates formation of a specific CENP-A/CENP-CCR complex. Lane 5: H3 nucleosome. Lanes 6-8: Increasing amounts of CENP-CCR are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-CCR complexes. C. Same experiment as in (B) using CENP-Cmotif. Lane 1. CENP-A nucleosome. Lanes 2-4. Increasing amounts of CENP-Cmotif are added to CENP-A nucleosome. Upon binding CENP-Cmotif, CENP-A nucleosome migrates slower through the gel. Note only modest change in mobility due to small size of CENP-Cmotif, comparing to CENP-CCR in (B). Lane 5. H3 nucleosome. Lanes 6-8. Increasing amounts of CENP-Cmotif are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-Cmotif complexes.


D . Native gel showing CENP - CCR competing out CENP - Cmotif bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome , Lane 2 : CENP - A / CENP - Cmotif complex . Lane 3 - 6 : Increasing amounts of CENP - CCR are added to the pre - formed CENP - A / CENP - Cmotif complex . Formation of slower migrating bands indicates that longer CENP - CCR is replacing shorter CENP - Cmotif bound to the CENP - A nucleosome .

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

D. Native gel showing CENP-CCR competing out CENP-Cmotif bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome, Lane 2: CENP-A/CENP-Cmotif complex. Lane 3-6: Increasing amounts of CENP-CCR are added to the pre-formed CENP-A/CENP-Cmotif complex. Formation of slower migrating bands indicates that longer CENP-CCR is replacing shorter CENP-Cmotif bound to the CENP-A nucleosome.


E . Native gel showing the inability of CENP - Cmotif to compete out CENP - CCR bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome . Lane 2 and 6 : CENP - CCR / CENP - A nucleosome complex . Lanes 3 - 5 : Increasing amounts of CENP - Cmotif are added to the pre - formed CENP - A / CENP - CCR complex . Formation of smear at high amounts of CENP - Cmotif added , indicates that CENP - Cmotif , at high concentrations , non - specifically binds CENP - A / CENP - CCR complex rather than replacing bound CENP - CCR . Lane 7 - 9 : Increasing amounts of CENP - CCR - short are added to the pre - formed CENP - A / CENP - CCR complex . Formation of bands with higher mobility indicates that smaller CENP - CCR - short is effectively replacing bigger CENP - CCR bound to CENP - A nucleosome .

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

E. Native gel showing the inability of CENP-Cmotif to compete out CENP-CCR bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome. Lane 2 and 6: CENP-CCR/CENP-A nucleosome complex. Lanes 3-5: Increasing amounts of CENP-Cmotif are added to the pre-formed CENP-A/CENP-CCR complex. Formation of smear at high amounts of CENP-Cmotif added, indicates that CENP-Cmotif, at high concentrations, non-specifically binds CENP-A/CENP-CCR complex rather than replacing bound CENP-CCR. Lane 7-9: Increasing amounts of CENP-CCR-short are added to the pre-formed CENP-A/CENP-CCR complex. Formation of bands with higher mobility indicates that smaller CENP-CCR-short is effectively replacing bigger CENP-CCR bound to CENP-A nucleosome.


G . Native gels showing the impaired ability of mutated CENP - CCR to form complexes with the CENP - A nucleosome . The molar ratio of CENP - CCR / CENP - A nucleosomes is shown above each lane .

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

G. Native gels showing the impaired ability of mutated CENP-CCR to form complexes with the CENP-A nucleosome. The molar ratio of CENP-CCR/CENP-A nucleosomes is shown above each lane.


A . Graph showing the relative abundance of undigested DNA ( 145 bp ) as a function of time during digestion with micrococcal nuclease ( MN - ase ) for the CENP - A nucleosome ( red ) , the CENP - A / CENP - CCR complex ( blue ) and the CENP - A / CENP - Cmotif complex ( black ) .

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

A. Graph showing the relative abundance of undigested DNA (145 bp) as a function of time during digestion with micrococcal nuclease (MN-ase) for the CENP-A nucleosome (red), the CENP-A/CENP-CCR complex (blue) and the CENP-A/CENP-Cmotif complex (black).


D . Same type of data as in ( A ) for the CENP - A nucleosome assembled with tailless H2A ( H2A , 1 - 109 ) alone ( pink ) and in complex with CENP - CCR ( light blue ) .

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

D. Same type of data as in (A) for the CENP-A nucleosome assembled with tailless H2A (H2A, 1-109) alone (pink) and in complex with CENP-CCR (light blue).


( A ) Representative single immunofluorescent staining for dsDNA at 6 h , 24 h , and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain . Scale bars , 5 μm .

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

(A) Representative single immunofluorescent staining for dsDNA at 6 h, 24 h, and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain. Scale bars, 5 μm.


( B ) Representative images of double - immunofluorescent stained for 53BP - 1 and dsDNA ( upper panel ) , Iba - 1 and dsDNA ( middle panel ) , and for GFAP and dsDNA ( lower panel ) . Scale bars , 5 μm .

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

(B) Representative images of double-immunofluorescent stained for 53BP-1 and dsDNA (upper panel), Iba-1 and dsDNA (middle panel), and for GFAP and dsDNA (lower panel). Scale bars, 5 μm.


( C ) Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mice brain following MCAO . ( D ) Quantitative analysis for Western blot analysis .

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

(C) Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mice brain following MCAO. (D) Quantitative analysis for Western blot analysis.


( E ) Immunohistochemical staining for cGAS at 3 d of reperfusion after tMCAO in peri - ischemic area as well as in the corresponding regions of sham control brains . Scale bars , 50 μm , 20 μm in insets . ( F ) Quantitative analysis of cGAS immunohistochemical staining . n = 6 mice per group . * * P < 0 . 01 , two - tailed unpaired Student ' s t test .

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

(E) Immunohistochemical staining for cGAS at 3 d of reperfusion after tMCAO in peri-ischemic area as well as in the corresponding regions of sham control brains. Scale bars, 50 μm, 20 μm in insets. (F) Quantitative analysis of cGAS immunohistochemical staining. n = 6 mice per group. **P<0.01, two-tailed unpaired Student's t test.


( G ) Representative double immunofluorescent stainings for cGAS ( red ) and 53BP1 ( green ) . n = 6 mice per group .

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

(G) Representative double immunofluorescent stainings for cGAS (red) and 53BP1 (green). n=6 mice per group.


Mice received daily intraperitoneal ( IP ) injections of A151 ( 300 μg ) or an equal volume of vehicle for 3 consecutive days after MCAO induction . ( A ) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT - PCR . n = 6 mice per group . * *

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

Mice received daily intraperitoneal (IP) injections of A151 (300 μg) or an equal volume of vehicle for 3 consecutive days after MCAO induction. (A) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT-PCR. n = 6 mice per group. **


( B ) The expression levels of cGAS , STING , and NF - κB proteins in the brains of indicated groups were measured using Western blot analysis . ( C ) Bar graph shows the relative protein expression levels of cGAS , STING , and NF - κB in the brains of indicated groups . n = 6 mice per group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The expression levels of cGAS, STING, and NF-κB proteins in the brains of indicated groups were measured using Western blot analysis. (C) Bar graph shows the relative protein expression levels of cGAS, STING, and NF-κB in the brains of indicated groups. n = 6 mice per group.


( D ) The mRNA expression levels of the AIM2 inflammasome components ( AIM2 , ASC , and caspase - 1 ) , effector of pyroptosis GSDMD , IL - 1β , and IL - 18 after MCAO were determined using qRT - PCR . n = 6 mice per group .

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

(D) The mRNA expression levels of the AIM2 inflammasome components (AIM2, ASC, and caspase-1), effector of pyroptosis GSDMD, IL-1β, and IL-18 after MCAO were determined using qRT-PCR. n = 6 mice per group.


( E ) The expression of AIM2 inflammasome components , GSDMD , and IL - 1β in the brains of indicated groups was measured using Western blot analysis . ( F ) Bar graph shows the relative protein expression levels of AIM2 inflammasome components , GSDMD , and IL - 1β in the brains of indicated groups . n = 6 mice per group .

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

(E) The expression of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups was measured using Western blot analysis. (F) Bar graph shows the relative protein expression levels of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups. n = 6 mice per group.


( A ) Representative images of immunohistochemical staining of IL - 1β , caspase - 1 , and GSDMD in the brains of indicated group . Scale bars , 50 μm . ( B ) Quantitative analysis for immunohistochemical staining . n = 6 mice per group .

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

(A) Representative images of immunohistochemical staining of IL-1β, caspase-1, and GSDMD in the brains of indicated group. Scale bars, 50 μm. (B) Quantitative analysis for immunohistochemical staining. n = 6 mice per group.


( C ) Representative images of double - immunofluorescent stained for GFAP and GSDMD , for Iba - 1 and GSDMD , and for NeuN and GSDMD in post - ischemic brains after tMCAO .

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

(C) Representative images of double-immunofluorescent stained for GFAP and GSDMD, for Iba-1 and GSDMD, and for NeuN and GSDMD in post-ischemic brains after tMCAO.


( D ) Representative images of double - immunofluorescent stained for Iba - 1 and GSDMD , for Iba - 1 and caspase - 1 , and for Iba - 1 and IL - 1β of sham + vehicle , MCAO + vehicle , and MCAO + A151 groups . Scale bars , 50 μm . ( E ) Bar graph shows the relative immunofluorescence intensity of GSDMD , caspase - 1 , and IL - 1β in the brains of indicated groups . n = 6 mice per group . DAPI staining : nuclei .

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

(D) Representative images of double-immunofluorescent stained for Iba-1 and GSDMD, for Iba-1 and caspase-1, and for Iba-1 and IL-1β of sham+vehicle, MCAO+vehicle, and MCAO+A151 groups. Scale bars, 50 μm. (E) Bar graph shows the relative immunofluorescence intensity of GSDMD, caspase-1, and IL-1β in the brains of indicated groups. n = 6 mice per group. DAPI staining: nuclei.


( A ) Expression of cGAS , STING , NF - κB , ASC , IL - 1β ( FL ) , caspase - 1 ( FL ) , and GSDMD in primary microglia lysates was detected using western blot . ( B ) Quantitative analysis for Western blot analysis of cGAS , STING , NF - κB , ASC , IL - 1β ( FL ) , caspase - 1 and GSDMD .

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

(A) Expression of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 (FL), and GSDMD in primary microglia lysates was detected using western blot. (B) Quantitative analysis for Western blot analysis of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 and GSDMD.


( C ) The protein levels of caspase - 1 p20 and IL - 1β ( p17 ) in the supernatant of cultured primary microglia were determined by western blot . ( D ) Quantitative analysis for Western blot analysis of caspapse - 1 and IL - 1β . * P < 0 . 05 , * * P < 0 . 01 compared with the untreated group , # P < 0 . 05 , # # P < 0 . 01 compared with the LPS + poly ( dA : dT ) group .

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

(C) The protein levels of caspase-1 p20 and IL-1β (p17) in the supernatant of cultured primary microglia were determined by western blot. (D) Quantitative analysis for Western blot analysis of caspapse-1 and IL-1β. *P < 0.05, **P<0.01 compared with the untreated group, #P < 0.05, ##P < 0.01 compared with the LPS+poly(dA:dT) group.


( E - F ) Immunofluorescence was conducted to detect the expression levels of cGAS , NF - κB , and pyroptosis associated molecules ( GSDMD , caspase - 1 , and IL - 1β ) in BV2 cells . n = 3 in each group . Scale bars , 50 μm .

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

(E-F) Immunofluorescence was conducted to detect the expression levels of cGAS, NF-κB, and pyroptosis associated molecules (GSDMD, caspase-1, and IL-1β) in BV2 cells. n = 3 in each group. Scale bars, 50 μm.


( A ) Gating strategy of brain - infiltrating immune cells including CD4 + T cells ( CD3 + CD4 + ) , CD8 + T cells ( CD3 + CD8 + ) , B cells ( CD3 - CD19 + ) , NK cells ( CD3 - NK1 . 1 + ) , monocyte / macrophages ( CD11b + CD45highF4 / 80 + ) , neutrophils ( CD11b + CD45highLy6G + ) , and microglia ( CD11b + CD45int ) and their expression of IL ( interleukin ) - 6 , TNF - α ( tumor necrosis factor - α ) , TGF - β ( transforming growth factor - β ) , and IL - 4 . FMO , fluorescence minus one . ( B ) Quantitative analysis of the counts of brain - infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment . ( C ) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment . n = 6 mice per group . ( D ) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment . n = 6 mice per group . *

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

(A) Gating strategy of brain-infiltrating immune cells including CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3-CD19+), NK cells (CD3-NK1.1+), monocyte/macrophages (CD11b+CD45highF4/80+), neutrophils (CD11b+CD45highLy6G+), and microglia (CD11b+CD45int) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-4. FMO, fluorescence minus one. (B) Quantitative analysis of the counts of brain-infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment. (C) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment. n = 6 mice per group. (D) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. *


( E ) Bar graph shows percentages of microglia expressing IL - 6 , TNF - α , TGF - β , and IL - 10 in the brains of MCAO mice receiving indicated treatment . n = 6 mice per group .

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

(E) Bar graph shows percentages of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group.


( F ) Representative images of immunofluorescent staining for ly6G in peri - infarct areas of MCAO mice receiving indicated treatment . ( G ) Bar graph shows the number of ly6G + neutrophils . n = 6 mice per group .

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

(F) Representative images of immunofluorescent staining for ly6G in peri-infarct areas of MCAO mice receiving indicated treatment. (G) Bar graph shows the number of ly6G+ neutrophils. n = 6 mice per group.


( H ) Representative images of immunofluorescent staining for Iba - 1 in peri - infarct areas of MCAO mice receiving indicated treatment . ( I ) Bar graph shows the number of Iba - 1 + microglia . n = 6 mice per group .

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

(H) Representative images of immunofluorescent staining for Iba-1 in peri-infarct areas of MCAO mice receiving indicated treatment. (I) Bar graph shows the number of Iba-1+ microglia. n = 6 mice per group.


( B ) Neurological tests were performed to evaluate the motor , sensory , and balance functions in mice receiving A151 , or vehicle at days 1 , 3 , 7 , and 14 after MCAO . n = 10 mice per group .

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

(B) Neurological tests were performed to evaluate the motor, sensory, and balance functions in mice receiving A151, or vehicle at days 1, 3, 7, and 14 after MCAO. n = 10 mice per group.


( C ) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO , the infarct area is shown in white .

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

(C) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO, the infarct area is shown in white.


( D ) Bar graph shows percentages of infarct volume of indicated groups . n = 10 mice per group . *

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

(D) Bar graph shows percentages of infarct volume of indicated groups. n = 10 mice per group. *


( E ) Flow cytometry plots and ( F ) summarized results show percentages of Annexin V + PI + cells in the brains of MCAO mice receiving A151 or vehicle . n = 6 mice per group .

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

(E) Flow cytometry plots and (F) summarized results show percentages of Annexin V+ PI+ cells in the brains of MCAO mice receiving A151 or vehicle. n = 6 mice per group.


( G ) Representative histology images for TUNEL staining . ( H ) Bar gragh shows percentages of TUNEL - positive cells . n = 6 mice per group .

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

(G) Representative histology images for TUNEL staining. (H) Bar gragh shows percentages of TUNEL-positive cells. n = 6 mice per group.


( A ) Representative double immunofluorescent stainings for Iba - 1 and cGAS , and for GFAP and cGAS . Scale bars , 10 μm .

(A) Representative double immunofluorescent stainings for Iba-1 and cGAS, and for GFAP and cGAS. Scale bars, 10 μm.


( D ) Representative genotyping analysis for WT , cGAS - KD , and cGAS - KO mice .

(D) Representative genotyping analysis for WT, cGAS-KD, and cGAS-KO mice.


( F ) Representative images of TTC staining of brain sections from vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , and A151 - treated cGAS - KO mice on day 3 after tMCAO . Three representative rostrocaudal levels of TTC staining are shown . ( G ) Bar graph shows percentages of infarct volume of indicated group . n = 6 mice per group . ( H ) Bar graph shows modified Neurological Severity Score ( mNSS ) in vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , and A151 - treated cGAS - KO mice n = 10 mice per group .

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

(F) Representative images of TTC staining of brain sections from vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice on day 3 after tMCAO. Three representative rostrocaudal levels of TTC staining are shown. (G) Bar graph shows percentages of infarct volume of indicated group. n = 6 mice per group. (H) Bar graph shows modified Neurological Severity Score (mNSS) in vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice n = 10 mice per group.


( D ) C / EBP β and AEP immunofluorescent staining of colons from age - matched healthy control and AD patients . White arrows indicate that C / EBP β and AEP were co - localized in AD patient ' s colon . ( E ) AEP and cleaved APP C586 fragments immunofluorescent staining of colons from age - matched healthy control and AD patient . White arrows indicate that AEP and cleaved APP C586 were co - localized in AD patient ' s colon . ( F ) AEP and cleaved Tau N368 fragments immunofluorescent staining of colons from age - matched healthy control and AD patient . Scale bar : 20 μm . White arrows indicate that APP C586 and AEP were co - localized in AD patient ' s colon .

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

(D) C/EBP β and AEP immunofluorescent staining of colons from age-matched healthy control and AD patients. White arrows indicate that C/EBP β and AEP were co-localized in AD patient's colon. (E) AEP and cleaved APP C586 fragments immunofluorescent staining of colons from age-matched healthy control and AD patient. White arrows indicate that AEP and cleaved APP C586 were co-localized in AD patient's colon. (F) AEP and cleaved Tau N368 fragments immunofluorescent staining of colons from age-matched healthy control and AD patient. Scale bar: 20 μm. White arrows indicate that APP C586 and AEP were co-localized in AD patient's colon.


( A ) Immunoblot showing p - C / EBP β , C / EBP β , AEP , APP , and Tau expression and proteolytic processing in mouse colon . ( B ) Immunoblot showing p - C / EBP β , C / EBP β , AEP , APP , and Tau expression and proteolytic processing in mouse brain .

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

(A) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse colon. (B) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse brain.


( B ) Quantitative analysis of AEP positive cells and C / EBPβ positive cells , respectively . The density of both AEP positive cells and C / EBPβ positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± SEM . * P = 0 . 0390 ( * P < 0 . 05 ) , * * * P = 0 . 0002 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( C ) Quantitative analysis of AT8 positive cells ( left ) and TauN368 positive cells ( right ) , respectively . The density of both AT8 positive cells and TauN368 positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . n = 5 in each group , data are shown as mean ± SEM . * P = 0 . 0194 ( * P < 0 . 05 ) , * * * P = 0 . 0001 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( D ) Quantitative analysis of Aβ positive cells ( left ) and APPC586 positive cells ( right ) , respectively . The fluorescence intensity of Aβ positive cells and the density APPC586 positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± SEM . * P = 0 . 0107 ( * P < 0 . 05 ) , * * P = 0 . 0048 ( 3xTg vs 3xTg AEP KO ) , * * P = 0 . 0044 ( 3xTg vs WT ) ( * * P < 0 . 01 ) , * * * P = 0 . 0006 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( E ) Quantitative analysis of AT8 positive cells ( upper ) and T22 positive cells ( lower ) , respectively . The fluorescence intensity of both AT8 positive cells and T22 positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± SEM . * * P = 0 . 0014 ( 3xTg vs 3xTg C / EBPβ + / - ) , * * P = 0 . 0030 ( 3xTg vs 3xTg AEP KO ) ( * * P < 0 . 01 ) , * * * P = 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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

(B) Quantitative analysis of AEP positive cells and C/EBPβ positive cells, respectively. The density of both AEP positive cells and C/EBPβ positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P =0.0390 (*P < 0.05), ***P =0.0002 (***P < 0.001), ****P < 0.0001, one-way ANOVA. (C) Quantitative analysis of AT8 positive cells (left) and TauN368 positive cells (right), respectively. The density of both AT8 positive cells and TauN368 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. n=5 in each group, data are shown as mean± SEM. *P =0.0194 (*P< 0.05), ***P =0.0001 (***P < 0.001), ****P < 0.0001, one-way ANOVA. (D) Quantitative analysis of Aβ positive cells (left) and APPC586 positive cells (right), respectively. The fluorescence intensity of Aβ positive cells and the density APPC586 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P=0.0107 (*P < 0.05), **P=0.0048 (3xTg vs 3xTg AEP KO), **P=0.0044 (3xTg vs WT) (**P< 0.01), ***P=0.0006 (***P< 0.001), ****P < 0.0001, one-way ANOVA. (E) Quantitative analysis of AT8 positive cells (upper) and T22 positive cells (lower), respectively. The fluorescence intensity of both AT8 positive cells and T22 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. **P=0.0014 (3xTg vs 3xTg C/EBPβ+/-), **P=0.0030 (3xTg vs 3xTg AEP KO) (**P<0.01), ***P=0.001, ****P<0.0001, one-way ANOVA.


( G ) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining . ( Scale bar : 5 μm ) . ( H ) Quantitative analysis of the synaptic densities in DSS - treated mice . Representative data of five samples , Data are shown as mean ± SEM . * * * * P < 0 . 0001 , one - way ANOVA . ( I ) Quantitative analysis of the spine density . Representative data of five samples , Data are shown as mean ± SEM . * * * * P < 0 . 0001 , one - way ANOVA .

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

(G) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm). (H) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA. (I) Quantitative analysis of the spine density. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA.


( E ) Gastrointestinal permeability barrier defect as determined by FITC - dextran translocation in pre - vagotomy DSS - treated , DSS - treated and vehicle - treated 3xTg mice . Data represent the mean ± SEM ; representative data of three samples ; * P = 0 . 0126 ( * P < 0 . 001 ) , * * * P = 0 . 0002 ( * * * P < 0 . 001 ) compared with control , one - way ANOVA .

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

(E) Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in pre-vagotomy DSS-treated, DSS-treated and vehicle-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P=0.0126 (*P < 0.001), ***P=0.0002 (***P < 0.001) compared with control, one-way ANOVA.


( F ) Pro - inflammatory cytokines IL - 6 , IL1 - β , and TNFα concentrations in colon lysates of vehicle - treated 3xTg mice , DSS - treated single side vagotomy 3xTg and DSS - treated 3xTg , respectively . Representative data of three samples ; data are shown as mean ± SEM . * P = 0 . 0372 ( * P < 0 . 05 ) , * * P = 0 . 0050 ( * * P < 0 . 01 ) , * * * P = 0 . 0008 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 compared with control , two - way ANOVA .

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

(F) Pro-inflammatory cytokines IL-6, IL1-β, and TNFα concentrations in colon lysates of vehicle-treated 3xTg mice, DSS-treated single side vagotomy 3xTg and DSS-treated 3xTg, respectively. Representative data of three samples; data are shown as mean ± SEM. *P=0.0372 (*P < 0.05), **P=0.0050 (**P < 0.01), ***P=0.0008 (***P <0.001), ****P<0.0001 compared with control, two-way ANOVA.


( H ) Representative electron microscopy of the synaptic structures in hippocampus . Red stars indicate the synapses . ( Scale bar : 1 μm ) . ( I ) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining . ( Scale bar : 5 μm ) . ( J ) Quantitative analysis of the synaptic densities in DSS - treated mice . Representative data of five samples , data are shown as mean ± SEM . * * * * P < 0 . 0001 , unpaired t tests . ( K ) Quantitative analysis of the spine density . Representative data of five samples , data are shown as mean ± SEM . * * * P = 0 . 0001 ( * * * P < 0 . 001 ) , unpair t tests .

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

(H) Representative electron microscopy of the synaptic structures in hippocampus. Red stars indicate the synapses. (Scale bar: 1 μm). (I) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm). (J) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, data are shown as mean ± SEM. ****P < 0.0001, unpaired t tests. (K) Quantitative analysis of the spine density. Representative data of five samples, data are shown as mean ± SEM. ***P =0.0001 (***P < 0.001), unpair t tests.


( A & B ) Cued and Contextual Fear - conditioning tests . Data represent the mean ± SEM of n = 6 - 8 mice per group ; * * P = 0 . 0067 ( 3xTg + DSS vs 3xTg ctrl ) , * * P = 0 . 0059 ( 3xTg + DSS vs 3xTg R - vagotomy + DSS ) ( * * P < 0 . 01 ) , * * * P = 0 . 0009 ( * * * P < 0 . 001 ) , two - way ANOVA ( A ) ; * * P = 0 . 0086 ( 3xTg + DSS vs 3xTg ctrl ) , * * P = 0 . 0085 ( 3xTg + DSS vs 3xTg R - vagotomy + DSS ) ( * * P < 0 . 01 ) , * * * P = 0 . 0005 ( * * * P < 0 . 001 ) , one - way ANOVA ( B ) .

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

(A&B) Cued and Contextual Fear-conditioning tests. Data represent the mean ± SEM of n = 6-8 mice per group; **P=0.0067 (3xTg+DSS vs 3xTg ctrl), **P=0.0059 (3xTg+DSS vs 3xTg R-vagotomy+DSS) (**P< 0.01), ***P=0.0009 (***P < 0.001), two-way ANOVA (A); **P=0.0086 (3xTg+DSS vs 3xTg ctrl), **P=0.0085 (3xTg+DSS vs 3xTg R-vagotomy+DSS) (**P< 0.01), ***P=0.0005 (***P < 0.001), one-way ANOVA (B).


( D ) Survival rate curve of the experimental mice during the experimental paradigm .

(D) Survival rate curve of the experimental mice during the experimental paradigm.


( B ) Immunofluorescent staining showed ATTO 550 - labeled Aβ PFFs and Tau N368 PFFs spread into different brain regions in WT mice 3 months after colonic injection . Scale bar : 20 μm .

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

(B) Immunofluorescent staining showed ATTO 550-labeled Aβ PFFs and Tau N368 PFFs spread into different brain regions in WT mice 3 months after colonic injection. Scale bar: 20 μm.


( C ) Immunohistochemistry staining of human Aβ propagating along the vagus nerve from WT mice colonic - injected with PBS , AD brain extracts or 5xFAD brain extracts , using human specific Aβ antibody ( BAN50 ) . Black arrows indicate the human Aβ in mouse vagus nerve . ( D ) Immunohistochemistry staining of human Tau propagating along the vagus nerve from WT mice colonic - injected with PBS or AD brain extracts , using human Tau specific antibody ( HT7 ) . Black arrows indicate the human Tau in mouse vagus nerve .

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

(C) Immunohistochemistry staining of human Aβ propagating along the vagus nerve from WT mice colonic-injected with PBS, AD brain extracts or 5xFAD brain extracts, using human specific Aβ antibody (BAN50). Black arrows indicate the human Aβ in mouse vagus nerve. (D) Immunohistochemistry staining of human Tau propagating along the vagus nerve from WT mice colonic-injected with PBS or AD brain extracts, using human Tau specific antibody (HT7). Black arrows indicate the human Tau in mouse vagus nerve.


( A ) Immunofluorescent staining of AEP and p - C / EBP β in cerebral cortex of brains of WT mice 6 months after colonic inoculation with either Aβ PFFs or Tau N368 PFFs . Scale bar : 20 μm . White arrows indicate that AEP and p - C / EBP β signals ( green ) were colocalized with ATTO 550 signals ( red ) , respectively .

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

(A) Immunofluorescent staining of AEP and p-C/EBP β in cerebral cortex of brains of WT mice 6 months after colonic inoculation with either Aβ PFFs or Tau N368 PFFs. Scale bar: 20 μm. White arrows indicate that AEP and p-C/EBP β signals (green) were colocalized with ATTO 550 signals (red), respectively.


( A ) Immunofluorescent staining of AEP ( red ) and C / EBP β ( green ) in CA1 region from hippocampus of brains from colonic - injected PBS , colonic - injected AD brain extracts , and pre - Vagotomy colonic injected AD brain extracts 3xTg mice . Scale bar : 20μm . ( B ) Quantitative analysis of AEP positive cells and C / EBP β positive cells , respectively . The density of both AEP and C / EBP β positive cells were significantly increased by colonic - injection of AD brain extracts and decreased through vagotomy before colonic - injection . Representative data of three samples , data are shown as mean ± SEM . * * P = 0 . 016 ( * * P < 0 . 01 ) , * * * * P < 0 . 0001 , one - way ANOVA .

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

(A) Immunofluorescent staining of AEP (red) and C/EBP β (green) in CA1 region from hippocampus of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. Scale bar: 20μm. (B) Quantitative analysis of AEP positive cells and C/EBP β positive cells, respectively. The density of both AEP and C/EBP β positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. **P =0.016 (**P < 0.01), ****P<0.0001, one-way ANOVA.


( C ) Immunofluorescent staining of cleaved APPC586 ( red ) and Aβ ( green ) in cerebral cortex of brains from colonic - injected PBS , colonic - injected AD brain extracts , and pre - Vagotomy colonic injected AD brain extracts 3xTg mice . ( D ) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells , respectively . The density of both cleaved APP C586 positive cells and Aβ positive cells were significantly increased by colonic - injection of AD brain extracts and decreased through vagotomy before colonic - injection . Representative data of three samples , data are shown as mean ± SEM . * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Immunofluorescent staining of cleaved APPC586 (red) and Aβ (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (D) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells, respectively. The density of both cleaved APP C586 positive cells and Aβ positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ****P<0.0001, one-way ANOVA.


( E ) Immunofluorescent staining of T22 ( red ) and HT7 ( green ) in cerebral cortex of brains from colonic - injected PBS , colonic - injected AD brain extracts , and pre - Vagotomy colonic injected AD brain extracts 3xTg mice . ( F ) Quantitative analysis of T22 positive cells . The density of T22 positive cells was significantly increased by colonic - injection of AD brain extracts and decreased through vagotomy before colonic - injection . Representative data of three samples , data are shown as mean ± SEM . * * * P = 0 . 0007 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA .

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(E) Immunofluorescent staining of T22 (red) and HT7 (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (F) Quantitative analysis of T22 positive cells. The density of T22 positive cells was significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ***P=0.0007 (***P < 0.001), ****P<0.0001, one-way ANOVA.


B , Total pup retrieval time in biological mothers ( n = 6 ) , foster mothers ( n = 7 ) , and virgin females ( n = 10 ) on 3 consecutive days .

B, Total pup retrieval time in biological mothers (n=6), foster mothers (n=7), and virgin females (n=10) on 3 consecutive days.


C , D Fraction of parental ( C ) and ignoring ( D ) behaviors of virgin females , of the total session duration in each of the three pup exposure days ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are displayed as individual measurements with the line connecting the mean value ; * p < 0 . 05 .

C,D Fraction of parental (C) and ignoring (D) behaviors of virgin females, of the total session duration in each of the three pup exposure days (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are displayed as individual measurements with the line connecting the mean value; *p < 0.05.


F , A representative image of a coronal brain section immunostained for c - Fos after pup retrieval on day 1 and day 3 . The mPFC region is indicated by dashed lines , which is also shown in the insets in higher magnification . Scale bar indicates 500 µm , 10 × magnification .

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F, A representative image of a coronal brain section immunostained for c-Fos after pup retrieval on day 1 and day 3. The mPFC region is indicated by dashed lines, which is also shown in the insets in higher magnification. Scale bar indicates 500 µm, 10× magnification.


G , Heat - map of the Z - score for the difference in the fraction of c - Fos expressing cells , between day 1 and day 3 of behavioral testing . Foster mothers ( n = 5 ) , virgins with pups ( n = 6 ) , virgins with object ( n = 7 ) .

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G, Heat-map of the Z-score for the difference in the fraction of c-Fos expressing cells, between day 1 and day 3 of behavioral testing. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7).


H , Representative coronal sections of the mPFC immunostained for c - Fos with the anatomical borders of the ACC indicated by dashed lines , in foster mothers ( FM ) and virgin females exposed to pups ( V pups ) or objects ( V object ) on day 1 and day 3 of behavioral testing . Scale bar 100 µm , 20 × magnification .

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H, Representative coronal sections of the mPFC immunostained for c-Fos with the anatomical borders of the ACC indicated by dashed lines, in foster mothers (FM) and virgin females exposed to pups (V pups) or objects (V object) on day 1 and day 3 of behavioral testing. Scale bar 100 µm, 20× magnification.


A , Reconstruction of the mouse brain following anterograde labeling with AAV - GFP in the ACC ( I ) ; representative coronal images showing site of injection and axonal distribution pattern in the dorsal striatum ( II ) and central lateral nucleus of the thalamus ( III , IV ) . Images in aI - aIV were taken from the Allen Mouse Brain Connectivity

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A, Reconstruction of the mouse brain following anterograde labeling with AAV-GFP in the ACC (I); representative coronal images showing site of injection and axonal distribution pattern in the dorsal striatum (II) and central lateral nucleus of the thalamus (III, IV). Images in aI-aIV were taken from the Allen Mouse Brain Connectivity Atlas


C , A representative coronal section showing labeling pattern at the site of injection . White arrows in the magnified image ( C , right ) point to double - labeled putative starter cells for transsynaptic retrograde labeling .

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C, A representative coronal section showing labeling pattern at the site of injection. White arrows in the magnified image (C, right) point to double-labeled putative starter cells for transsynaptic retrograde labeling.


D , Representative coronal sections showing bidirectional connectivity patterns with the thalamus ( I ) and monosynaptic input to the ACC from the claustrum ( II ) and ventral CA1 ( III ) . Scale bars 500 µm , 10 × magnification ( left images ) and 100 µm , 20 × magnification ( right images ) .

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D, Representative coronal sections showing bidirectional connectivity patterns with the thalamus (I) and monosynaptic input to the ACC from the claustrum (II) and ventral CA1 (III). Scale bars 500 µm, 10× magnification (left images) and 100 µm, 20× magnification (right images).


G , Representative coronal sections of the dorsal thalamus , following c - Fos immunolabeling on day 1 ( left ) and day 3 ( right ) , with the ILM subnuclei indicated ; CM central medial nucleus , CL central lateral nucleus , PCN paracentral nucleus , RH rhomboid nucleus . Scale bar 200 µm , 10 × magnification . Foster mothers ( n = 5 ) , virgins with pups ( n = 6 ) , virgins with object ( n = 7 ) .

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G, Representative coronal sections of the dorsal thalamus, following c-Fos immunolabeling on day 1 (left) and day 3 (right), with the ILM subnuclei indicated; CM central medial nucleus, CL central lateral nucleus, PCN paracentral nucleus, RH rhomboid nucleus. Scale bar 200 µm, 10× magnification. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7).


H , Fraction of c - Fos + cells in different ILM subnuclei , virgin exposed to pups ( left ) , in foster mothers ( middle ) , and virgin exposed to object ( right ) , on day 1 and day 3 of behavioral testing . Foster mothers ( n = 5 ) , virgins with pups ( n = 6 ) , virgins with object ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are expressed as mean ± s . e . m ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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H, Fraction of c-Fos+ cells in different ILM subnuclei, virgin exposed to pups (left), in foster mothers (middle), and virgin exposed to object (right), on day 1 and day 3 of behavioral testing. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are expressed as mean ± s.e.m; *p < 0.05, **p < 0.01, ***p < 0.001.


B , Representative coronal sections of the PFC , following the labeling schemes described in ( A ) , for mapping inputs onto ACC excitatory ( B , top ) and inhibitory ( B , bottom ) neurons . High magnification images ( B , right top and bottom ) show the double - labeled , putative starter cells for transsynaptic retrograde labeling . Scale bars represent 200 µm , 10 × magnification ( left top and bottom ) , and 50 µm ( right top and bottom ) , 20 × magnification .

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B, Representative coronal sections of the PFC, following the labeling schemes described in (A), for mapping inputs onto ACC excitatory (B, top) and inhibitory (B, bottom) neurons. High magnification images (B, right top and bottom) show the double-labeled, putative starter cells for transsynaptic retrograde labeling. Scale bars represent 200 µm, 10× magnification (left top and bottom), and 50 µm (right top and bottom), 20× magnification.


C , Representative coronal images of the thalamus , immunolabeled for c - Fos , following retrograde labeling from excitatory ( C , top ) or inhibitory ( C , bottom ) neurons in the ACC . White arrows in high magnification images indicate colocalization of c - Fos and td - Tomato ( C , right top and bottom ) . Scale bars represent 200 µm , 10 × magnification ( left top and bottom ) , and 25 µm ( right top and bottom ) , 20 × magnification .

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C, Representative coronal images of the thalamus, immunolabeled for c-Fos, following retrograde labeling from excitatory (C, top) or inhibitory (C, bottom) neurons in the ACC. White arrows in high magnification images indicate colocalization of c-Fos and td-Tomato (C, right top and bottom). Scale bars represent 200 µm, 10× magnification (left top and bottom), and 25 µm (right top and bottom), 20× magnification.


D , Quantification of the number of neurons in CL region projecting to excitatory or inhibitory ACC neurons , as a fraction of the total thalamic contribution ( top ) and fraction of neurons co - expressing tdTomato and c - Fos of the total tdTomato + population in CL ( bottom ) . Foster mothers ( n = 3 ) , virgins with pups ( n = 4 ) , virgins with object ( n = 4 ) . Data were analyzed using student ' s t - test and are displayed as mean ± s . e . m ;

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D, Quantification of the number of neurons in CL region projecting to excitatory or inhibitory ACC neurons, as a fraction of the total thalamic contribution (top) and fraction of neurons co-expressing tdTomato and c-Fos of the total tdTomato+ population in CL (bottom). Foster mothers (n=3), virgins with pups (n=4), virgins with object (n=4). Data were analyzed using student's t-test and are displayed as mean ± s.e.m;


, Representative in - situ hybridization images of Gal in the ILM taken from the Allen Mouse Brain Atlas representative coronal images showing the expression of Gal neurons in Gal - Cre : : FLEX - YFP female mice ( bottom right and left ) .

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, Representative in-situ hybridization images of Gal in the ILM taken from the Allen Mouse Brain Atlas representative coronal images showing the expression of Gal neurons in Gal-Cre::FLEX-YFP female mice (bottom right and left).


, Representative image of the ILM immunolabeled for GFP and c - Fos , following pup retrieval test . Yellow and white arrows indicate c - Fos expressing Gal - positive and Gal - negative neurons , respectively . Scale bars represent 100 µm , 20 × magnification .

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, Representative image of the ILM immunolabeled for GFP and c-Fos, following pup retrieval test. Yellow and white arrows indicate c-Fos expressing Gal-positive and Gal-negative neurons, respectively. Scale bars represent 100 µm, 20× magnification.


G , Graph plots showing the fraction of c - Fos expressing Gal - positive ( Galc - Fos + ) and Gal - negative neurons ( DAPIc - Fos + ) in the CL , PCN , and CM after pup retrieval test ( G , top ) or object exposure ( G , bottom ) . Data were analyzed using 2 - way ANOVA and expressed as mean ± s . e . m . ; n = 3 ; * p < 0 . 05 .

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G, Graph plots showing the fraction of c-Fos expressing Gal-positive (Galc-Fos+) and Gal-negative neurons (DAPIc-Fos+) in the CL, PCN, and CM after pup retrieval test (G, top) or object exposure (G, bottom). Data were analyzed using 2-way ANOVA and expressed as mean ± s.e.m.; n=3; *p < 0.05.


B , C , Quantification of pup retrieval latencies following ACC chemogenetic activation with hM3D ( n = 6 ) ( B ) or chemogenetic inhibition using hM4D ( n = 7 ) ( C ) compared to control groups in virgin females ( n = 8 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are displayed as mean ± s . e . m * p < 0 . 05 , * * p < 0 . 01 . D , Same as ( C ) but for mothers ( n = 7 )

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B,C, Quantification of pup retrieval latencies following ACC chemogenetic activation with hM3D (n=6) (B) or chemogenetic inhibition using hM4D (n=7) (C) compared to control groups in virgin females (n=8). Data were analyzed using 2-way ANOVA with repeated measures and are displayed as mean ± s.e.m* p< 0.05, ** p< 0.01. D, Same as (C) but for mothers (n=7)


E , Representative images showing AAV - DIO - CAG - hM3D - 2A - tdTomato and c - Fos expression in the PFC of virgin females , 2 hours following CNO / vehicle administration . Masks demonstrating distribution of DAPI + c - Fos ( green ) , DAPI + dTomato ( magenta ) and their colocalization ( white ) . Scale bars 100 µm , 20 × magnification .

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E, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the PFC of virgin females, 2 hours following CNO/vehicle administration. Masks demonstrating distribution of DAPI + c-Fos (green), DAPI + dTomato (magenta) and their colocalization (white). Scale bars 100 µm, 20× magnification.


F , Fraction c - Fos + cells of the total DAPI ( top ) or hM3D - 2A - dTomato ( bottom ) population in the ACC , following CNO / vehicle administration . ( n = 3 ) . Data were analyzed using student ' s t - test and are displayed as mean ± s . e . m ; * p < 0 . 05 , * * p < 0 . 01 .

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F, Fraction c-Fos+ cells of the total DAPI (top) or hM3D-2A-dTomato (bottom) population in the ACC, following CNO/vehicle administration. (n=3). Data were analyzed using student's t-test and are displayed as mean ± s.e.m; * p< 0.05, ** p< 0.01.


G , Representative images of the CL , immunolabeled for c - Fos , following hM3D - mediated neuronal activation of the ACC . Scale bars 200 µm , 20 × magnification .

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G, Representative images of the CL, immunolabeled for c-Fos, following hM3D-mediated neuronal activation of the ACC. Scale bars 200 µm, 20× magnification.


H , Spearman correlation between the hM3D - mediated activation of the ACC ( as measured in ( F , top ) ) and the CL region .

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H, Spearman correlation between the hM3D-mediated activation of the ACC (as measured in (F, top)) and the CL region.


B , Representative images showing AAV - DIO - CAG - hM3D - 2A - tdTomato and c - Fos expression in the CL , 2 h after CNO administration . Scale bars 200 µm , 10 × magnification ( left ) and 50 µm , 20 × magnification ( right ) .

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B, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the CL, 2 h after CNO administration. Scale bars 200 µm, 10× magnification (left) and 50 µm, 20× magnification (right).


C , Quantification of pup retrieval latencies in virgin females following CLGal + chemogenetic activation with hM3D compared to GFP - expressing control group ( n = 7 ) . Data were analyzed using 2 - way ANOVA with repeated measures and are presented as mean ± s . e . m ; * p < 0 . 05 .

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

C, Quantification of pup retrieval latencies in virgin females following CLGal+ chemogenetic activation with hM3D compared to GFP-expressing control group (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are presented as mean ± s.e.m; * p < 0.05.


HeLa cells were uninfected ( ' control ' ) or infected with B . abortus ( ' B . abortus ' ) . Cell lysates were collected at the indicated time points and analyzed by Western blotting . ( A ) Representative immunoblots for pIRE1 , spliced - XBP1 , and GAPDH . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software . ( B , C ) Relative protein levels of pIRE1 ( B ) and spliced - XBP1 ( C ) in uninfected control ( open circles ) and Brucella - infected ( solid circles ) cells . The protein levels at time 0 hr were assigned the value 1 . Data are means ± SD from three independent experiments .

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HeLa cells were uninfected ('control') or infected with B. abortus ('B. abortus'). Cell lysates were collected at the indicated time points and analyzed by Western blotting. (A) Representative immunoblots for pIRE1, spliced-XBP1, and GAPDH. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software. (B, C) Relative protein levels of pIRE1 (B) and spliced-XBP1 (C) in uninfected control (open circles) and Brucella-infected (solid circles) cells. The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.


( D ) Representative immunoblots for pPERK , cleaved - ATF6 , and GAPDH , and relative protein levels of pPERK and cleaved - ATF6 in control ( open bars ) and Brucella - infected ( solid bars ) cells . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software , and the results are shown in the bar graphs . As a positive control for activation of PERK or ATF6 , HeLa cells were treated with 5 μg / ml of tunicamycin for 8hr ( ' Tm ' ) . The protein levels at time 0 hr were assigned the value 1 . Data are means ± SD from three independent experiments .

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(D) Representative immunoblots for pPERK, cleaved-ATF6, and GAPDH, and relative protein levels of pPERK and cleaved-ATF6 in control (open bars) and Brucella-infected (solid bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. As a positive control for activation of PERK or ATF6, HeLa cells were treated with 5 μg/ml of tunicamycin for 8hr ('Tm'). The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.


( E ) Representative immunoblots for Sar1 , Sec23 , Sec24D , and GAPDH , and relative protein levels of Sar1 , Sec23 , and Sec24D in control ( open bars ) and Brucella - infected ( solid bars ) cells . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software , and the results are shown in the bar graphs . The protein levels in control cells were assigned the value 1 . Data are means ± SD from three independent experiments . * : p < 0 . 05 ; * * : p < 0 . 01 .

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(E) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (open bars) and Brucella-infected (solid bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. The protein levels in control cells were assigned the value 1. Data are means ± SD from three independent experiments. *: p<0.05; **: p<0.01.


( A ) Representative immunoblots showing co - immunoprecipitation with pIRE1 . After 5 hr of Tm treatment , immunoprecipitation was performed on HeLa cell lysates with an anti - pIRE1 antibody ( lanes labeled ' pIRE1 ' ) or control anti - rabbit IgG ( lanes labeled ' IgG ' ) , and the immunoprecipitates were analyzed by Western blotting with a panel of antibodies against molecules involved in the ER - Golgi vesicular transport pathways or against ER - ( Sec61α , HSP47 , and calnexin ) , ERGIC - ( ERGIC53 ) and cis - Golgi - ( GM130 ) resident proteins .

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(A) Representative immunoblots showing co-immunoprecipitation with pIRE1. After 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-pIRE1 antibody (lanes labeled 'pIRE1') or control anti-rabbit IgG (lanes labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with a panel of antibodies against molecules involved in the ER-Golgi vesicular transport pathways or against ER- (Sec61α, HSP47, and calnexin), ERGIC- (ERGIC53) and cis-Golgi- (GM130) resident proteins.


( B ) Representative immunoblot showing the co - immunoprecipitation of pIRE1 with Yip1A . Following 5 hr of Tm treatment , immunoprecipitation was performed on HeLa cell lysates with an anti - Yip1A antibody ( lane labeled ' Yip1A ' ) or control anti - rabbit IgG ( lane labeled ' IgG ' ) , and the immunoprecipitates were analyzed by Western blotting with an anti - pIRE1 antibody .

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(B) Representative immunoblot showing the co-immunoprecipitation of pIRE1 with Yip1A. Following 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-Yip1A antibody (lane labeled 'Yip1A') or control anti-rabbit IgG (lane labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with an anti-pIRE1 antibody.