B . Impaired recruitment of GFP - LC3 on MVP positive Listeria . HeLa were transfected with MVP - tomato ( red ) GFP - LC3 ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for microscopy , stained with phalloidin ( blue ) . Inset regions are magnified . scale bar represents 1 µm . MVP / or actin positive were never recognized by GFP - LC3 . Arrows point to at different steps of infection process : 1 ) InlK over - expressing bacterium is totally covered by MVP ; 2 ) bacterium is partially labeled with MVP ( at poles ) actin ( at center ) ; 3 ) bacterium is completely covered by actin ; 4 ) bacterium is enclosed in an GFP - LC3 positive autophagosome .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Impaired recruitment of GFP-LC3 on MVP positive Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue). Inset regions are magnified. The scale bar represents 1 µm. MVP and/or actin positive bacteria were never recognized by GFP-LC3. Arrows point to bacteria at different steps of the infection process: 1) InlK over-expressing bacterium is totally covered by MVP; 2) bacterium is partially labeled with MVP (at the poles) and actin (at the center); 3) bacterium is completely covered by actin; 4) bacterium is enclosed in an GFP-LC3 positive autophagosome.


C . Kinetics of autophagy escape for MVP positive Listeria . Jeg3 were transfected with MVP - tomato ( red ) GFP - LC3 ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , prepared for real - time video microscopy . Image series were collected every 5 min for 2 h . Time is indicated along Y axis . left panel shows that GFP - LC3 membranous aggregate detaches from MVP positive Listeria . entire image can be viewed as Video S2 . right panel shows that GFP - LC3 membranous aggregate on MVP positive does not lead to an autophagosome formation , whereas those efficiently divided . entire image can be viewed as Video S3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Kinetics of autophagy escape for MVP positive Listeria. Jeg3 cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, and prepared for real-time video microscopy. Image series were collected every 5 min for 2 h. Time is indicated along the Y axis. The left panel shows that the GFP-LC3 membranous aggregate detaches from the MVP positive Listeria. The entire image sequence can be viewed as Video S2. The right panel shows that the GFP-LC3 membranous aggregate on MVP positive bacteria does not lead to an autophagosome formation, whereas those bacteria efficiently divided. The entire image sequence can be viewed as Video S3.


D . Impaired recruitment of GFP - LC3 ubiquitin to MVP positive ΔactA Listeria . HeLa were transfected with MVP - tomato ( red ) GFP - LC3 ( green ) , infected with InlK over - expressing ΔactA ( ΔactA + pADc - inlK ) for 4 h , fixed for microscopy , stained with anti - ubiquitin antibody ( blue ) DAPI ( ) . Inset regions are magnified . scale bar represents 1 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

D. Impaired recruitment of GFP-LC3 and ubiquitin to MVP positive ΔactA Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing ΔactA (ΔactA+pADc-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with anti-ubiquitin antibody (blue) and DAPI (white). Inset regions are magnified. The scale bar represents 1 µm.


E . LC3 in infected RAW 267 . 4 macrophages . Left panel : RAW 267 . 4 macrophages were infected with L . monocytogenes EGD ( WT ) , ΔactA or ΔactA + InlK for 6 h . total lysates were immunobloted for LC3 actin . Western blot is representative from 3 independent experiments . Right panel : of relative LC3 - II level ( mean ± ) shown in left panel . Statistical analyses were performed on results of 3 independent experiments using Student ' s t . P values of < 0 . 05 were considered statistically different .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. LC3 levels in infected RAW 267.4 macrophages. Left panel: RAW 267.4 macrophages were infected with L. monocytogenes EGD (WT), ΔactA or ΔactA+InlK for 6 h. Cell total lysates were immunobloted for LC3 and actin. Western blot is representative from 3 independent experiments. Right panel: Quantification of the relative LC3-II level (mean ± SEM) shown in the left panel. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different.


A . InlK ActA expression in Listeria strains used for survival assays . Total lysates of L . monocytogenes EGD - ( pADc - GFP ) , EGD - ( pADc - inlK ) , ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) grown in BHI were immunoblotted using anti - ActA anti - InlK antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. InlK and ActA expression in Listeria strains used for survival assays. Total lysates of L. monocytogenes EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) grown in BHI were immunoblotted using anti-ActA and anti-InlK antibodies.


B . Intracellular survival of EGD - ( pADc - GFP ) , EGD - ( pADc - inlK ) , ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) in RAW 267 . 4 macrophages . Statistical analyses were performed on results of 3 independent experiments using Student t . P values of < 0 . 05 were considered statistically different are labeled here as * .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Intracellular survival of EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in RAW 267.4 macrophages. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.


C . Intracellular survival of ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) in MVP - GFP transfected Jeg3 . Statistical analyses were performed on results of 3 independent experiments using Student ' s t . P values of < 0 . 05 were considered statistically different are labeled here as * .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP-GFP transfected Jeg3 cells. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different and are labeled here as *.


D . Intracellular survival of ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) in MVP - GFP transfected HeLa . Statistical analyses were performed on results of 3 independent experiments using Student ' s t . P values of < 0 . 05 were considered statistically different are labeled here as * .

D. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP-GFP transfected HeLa cells. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different and are labeled here as *.


E . MVP in RAW 267 . 4 macrophages treated with MVP - siRNA . Western blot is representative from 3 independent experiments .

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

E. MVP levels in RAW 267.4 macrophages treated with MVP-siRNA. Western blot is representative from 3 independent experiments.


F . Intracellular survival of ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) in MVP knock - down RAW 267 . 4 macrophages . Statistical analyses were performed on results of 3 independent experiments using Student t . P values of < 0 . 05 were considered statistically different are labeled here as * .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP knock-down RAW 267.4 macrophages. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.


A Double - plotted representative actograms of indicated genotypes . Flies are monitored in LD for 4 days then DD for 7 days . Dicer2 ( dcr2 ) is co - expressed to enhance effects of RNAi . boxes indicate phase black boxes indicate phase . Gray shades indicate DD . G4 , GAL4 ; U , UAS .

A Double-plotted representative actograms of the indicated genotypes. Flies are monitored in LD for 4 days and then DD for 7 days. Dicer2 (dcr2) is co-expressed to enhance the effects of RNAi. White boxes indicate the light phase and black boxes indicate the dark phase. Gray shades indicate DD. G4, GAL4; U, UAS.


B - D period of DD locomotor rhythms of flies with Nipped - A knocked down controls . Data information : ( B - H ) Error bars represent standard error of mean ( ) . Digits on bars are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using One - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # P < 0 . 001 , * compared with GAL4 controls ; # compared with UAS controls . bar indicates UAS or GAL4 controls . Gray bar indicates flies with Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D The period of DD locomotor rhythms of flies with Nipped-A knocked down and controls. Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.


E , F Using - sensitive GAL80 system to control Nipped - A RNAi expression . ( E ) period of DD locomotor rhythms of flies raised at 18ºC tested at 29ºC . ( F ) period of DD locomotor rhythms of flies raised at 29ºC tested at 18ºC . Data information : ( B - H ) Error bars represent standard error of mean ( ) . Digits on bars are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using One - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # P < 0 . 001 , * compared with GAL4 controls ; # compared with UAS controls . bar indicates UAS or GAL4 controls . Gray bar indicates flies with Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Using temperature-sensitive GAL80 system to control Nipped-A RNAi expression. (E) The period of DD locomotor rhythms of flies raised at 18ºC and tested at 29ºC. (F) The period of DD locomotor rhythms of flies raised at 29ºC and tested at 18ºC. Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.


G , H period of DD locomotor rhythms of flies treated with RU486 to activate pdfG4 - geneswitch ( pdf - GS ) driver ( G ) or vehicle control ( H ) . Data information : ( B - H ) Error bars represent standard error of mean ( ) . Digits on bars are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using One - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # P < 0 . 001 , * compared with GAL4 controls ; # compared with UAS controls . bar indicates UAS or GAL4 controls . Gray bar indicates flies with Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H The period of DD locomotor rhythms of flies treated with RU486 to activate the pdfG4-geneswitch (pdf-GS) driver (G) or vehicle control (H). Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.


A Plots of relative mRNA abundance vs circadian time ( CT ) for clock genes determined by qRT - PCR in whole head extracts of Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 / + ) control ( timG4 / + ; Udcr2 / + ) collected on DD1 ( tim / cry , n = 5 ; Pdp1ε / per / clk / cyc , n = 3 ; vri , n = 6 ) . For each time series , value of lowest time point was set to 1 .

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

A Plots of relative mRNA abundance vs circadian time (CT) for clock genes determined by qRT-PCR in whole head extracts of Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1/+) and control (timG4/+;Udcr2/+) collected on DD1 (tim/cry, n=5; Pdp1ε/per/clk/cyc, n=3; vri, n=6). For each time series, the value of the lowest time point was set to 1.


B Western blots of from whole head extracts of Nipped - A RNAi control flies collected on DD1 probed with TIM , PDP1ε PER antibodies . C of TIM ( n = 5 ) , PDP1ε ( n = 3 ) PER ( n = 3 ) of blots in ( B ) . TIM , PDP1ε PER were normalized to that of HSP70 . For each time series , value of control at peak time point was set to 1 .

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

B Western blots of proteins from whole head extracts of Nipped-A RNAi and control flies collected on DD1 and probed with TIM, PDP1ε and PER antibodies. C Quantification of TIM (n=5), PDP1ε (n=3) and PER (n=3) protein levels of blots in (B). TIM, PDP1ε and PER protein levels were normalized to that of HSP70. For each time series, the value of the control at the peak time point was set to 1.


D Brains from Udcr2 / + ; cryG4 - 16 / + Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi flies collected at CT0 , 4 , 8 , 12 , 16 , 20 on DD1 were immunostained with TIM ( green ) , PDP1ε ( red ) , PER ( green ) PDF ( red or green ) antisera . scale bar represents 10µm . E of TIM , PDP1ε PER in s - LNvs of in ( D ) ( TIM : CT8 : Udcr2 / + ; cryG4 - 16 / + , n = 89 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 58 ; CT20 : Udcr2 / + ; cryG4 - 16 / + , n = 130 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 80 ; PDP1ε : CT4 : Udcr2 / + ; cryG4 - 16 / + , n = 46 ; Udcr2 / + ; cryG4 - 16 / UNipped - RNAi , n = 26 ; CT16 : Udcr2 / + ; cryG4 - 16 / + , n = 65 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 41 ; PER : CT0 : Udcr2 / + ; cryG4 - 16 / + , n = 90 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 58 ; CT12 : Udcr2 / + ; cryG4 - 16 / + , n = 86 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 64 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Brains from Udcr2/+;cryG4-16/+ and Udcr2/+;cryG4-16/UNipped-ARNAi flies collected at CT0, 4, 8, 12, 16, 20 on DD1 were immunostained with TIM (green) , PDP1ε (red), PER (green) and PDF (red or green) antisera. The scale bar represents 10µm. E Quantification of TIM, PDP1ε and PER protein levels in the s-LNvs of images in (D) (TIM: CT8: Udcr2/+;cryG4-16/+, n=89; Udcr2/+;cryG4-16/UNipped-ARNAi, n=58; CT20: Udcr2/+;cryG4-16/+, n=130; Udcr2/+;cryG4-16/UNipped-ARNAi, n=80; PDP1ε: CT4: Udcr2/+;cryG4-16/+, n=46; Udcr2/+;cryG4-16/UNipped- RNAi, n=26; CT16: Udcr2/+;cryG4-16/+, n=65; Udcr2/+;cryG4-16/UNipped-ARNAi, n=41; PER: CT0: Udcr2/+;cryG4-16/+, n=90; Udcr2/+;cryG4-16/UNipped-ARNAi, n=58; CT12: Udcr2/+;cryG4-16/+, n=86; Udcr2/+;cryG4-16/UNipped-ARNAi, n=64).


A , B period of DD locomotor rhythm of Nipped - A RNAi flies over - expressing tim ( A ) or carrying heterozygous tim01 mutation ( B ) . Ptim is a tim cDNA construct driven by tim promoter . Data information : Error bars represent . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with over - expression or mutant flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated clock gene . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated clock gene Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing tim (A) or carrying heterozygous tim01 mutation (B). Ptim is a tim cDNA construct driven by tim promoter. Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.


C , D period of DD locomotor rhythm of Nipped - A RNAi flies over - expressing Pdp1ε ( C ) or carrying heterozygous Pdp1ε3135 mutation ( D ) . Data information : Error bars represent . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with over - expression or mutant flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated clock gene . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated clock gene Nipped - A knocked down . G4 , GAL4 ; U , UAS .

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

C, D The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing Pdp1ε (C) or carrying heterozygous Pdp1ε3135 mutation (D). Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.


E , F period of DD locomotor rhythm of Nipped - A RNAi flies over - expressing per ( E ) or carrying perL mutation ( F ) . Data information : Error bars represent . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with over - expression or mutant flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated clock gene . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated clock gene Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing per (E) or carrying perL mutation (F). Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.


A Plots of relative mRNA abundance of Nipped - A in whole head extracts of w1118 flies in LD DD1 determined by qRT - PCR ( n = 3 ) .

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

A Plots of relative mRNA abundance of Nipped-A in whole head extracts of w1118 flies in LD and DD1 determined by qRT-PCR (n=3).


B Left panel : Western blots of from whole heads of w1118 Nipped - A RNAi flies collected during LD or DD1 probed with NIPPED - A antibody . Right panel : of NIPPED - A of blots in left panel ( LD , n = 3 ; DD1 , n = 5 ) .

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

B Left panel: Western blots of proteins from whole heads of w1118 and Nipped-A RNAi flies collected during LD or DD1 and probed with NIPPED-A antibody. Right panel: quantification of NIPPED-A protein levels of blots in the left panel (LD, n=3; DD1, n=5).


C immunoprecipitation ( ChIP ) assays to detect NIPPED - A binding at E - box , transcription start site ( TSS ) gene of tim , Pdp1ε per using w1118 flies ( n = 3 ) . Data information : Error bars represent . Two - way ANOVA , significant effect between NIPPED - A IgG were found for tim E - box ( P < 0 . 001 ) , Pdp1ε E - box ( P < 0 . 001 ) , per E - box ( P < 0 . 001 ) , tim TSS ( P < 0 . 001 ) , Pdp1ε TSS ( P < 0 . 001 ) , per TSS ( P < 0 . 001 ) , tim gene ( P < 0 . 001 ) , Pdp1ε gene per gene ( P < 0 . 001 ) . Student ' s t - , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . G4 , GAL4 ; U , UAS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromatin immunoprecipitation (ChIP) assays to detect NIPPED-A binding at E-box, transcription start site (TSS) and gene body of tim, Pdp1ε and per using w1118 flies (n=3). Data information: Error bars represent SEM. Two-way ANOVA, significant effect between NIPPED-A and IgG were found for tim E-box (P<0.001), Pdp1ε E-box (P<0.001), per E-box (P<0.001), tim TSS (P<0.001), Pdp1ε TSS (P<0.001), per TSS (P<0.001), tim gene body (P<0.001), Pdp1ε gene body and per gene body (P<0.001). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. G4, GAL4; U, UAS.


A Plots of relative pre - mRNA abundance of tim ( n = 5 ) Pdp1ε ( n = 3 ) determined by qRT - PCR in whole head extracts of Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 ) control ( timG4 / + ; Udcr2 / + ) flies during DD1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Plots of relative pre-mRNA abundance of tim (n=5) and Pdp1ε (n=3) determined by qRT-PCR in the whole head extracts of Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) flies during DD1.


B ChIP assays to detect ub - H2B binding at E - box , TSS gene of tim , Pdp1ε , per clk in Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 ) control ( timG4 / + ; Udcr2 / + ) ( n ≥ 3 ) . Data information : Student ' s t - , * P < 0 . 05 . Two - way ANOVA , significant effect between ub - H2B IgG were found for genomic regions tested ( P < 0 . 001 ) . Significant effect of genotypes were found for tim E - box ( P < 0 . 05 ) , Pdp1ε E - box ( P < 0 . 01 ) , tim TSS ( P < 0 . 01 ) Pdp1ε TSS ( P < 0 . 001 ) .

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

B ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim, Pdp1ε, per and clk in Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) (n≥3). Data information: Student's t-test, *P < 0.05. Two-way ANOVA, significant effect between ub-H2B and IgG were found for all genomic regions tested (P<0.001). Significant effect of genotypes were found for tim E-box (P<0.05), Pdp1ε E-box (P<0.01), tim TSS (P<0.01) and Pdp1ε TSS (P<0.001).


A , B period ( A ) power ( B ) of DD locomotor rhythms of flies with not knocked down controls . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with not overexpression flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not Nipped - A knocked down .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The period (A) and power (B) of DD locomotor rhythms of flies with not knocked down and controls. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


C Plots of relative mRNA abundance vs CT for clock genes determined by qRT - PCR in whole head extracts of not RNAi ( timG4 / + ; Udcr2 / UnotRNAi ) control ( timG4 / + ; Udcr2 / + ) collected on DD1 ( n = 5 ) .

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

C Plots of relative mRNA abundance vs CT for clock genes determined by qRT-PCR in whole head extracts of not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) collected on DD1 (n=5).


D ChIP assays to detect ub - H2B binding at E - box , TSS gene of tim Pdp1ε in not RNAi ( timG4 / + ; Udcr2 / UnotRNAi ) control ( timG4 / + ; Udcr2 / + ) ( n = 3 ) . Student ' s t - , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Statistical difference is measured using two - way ANOVA , P < 0 . 001 , significant effect of genotypes were found for tim E - box ( P < 0 . 01 ) , Pdp1ε E - box ( P < 0 . 05 ) , tim TSS ( P < 0 . 05 ) Pdp1ε TSS ( P < 0 . 001 ) tim gene ( P < 0 . 01 ) . G4 , GAL4 ; U , UAS .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim and Pdp1ε in not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) (n=3). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical difference is measured using two-way ANOVA, P<0.001, significant effect of genotypes were found for tim E-box (P<0.01), Pdp1ε E-box (P<0.05), tim TSS (P<0.05) and Pdp1ε TSS (P<0.001) and tim gene body (P<0.01). G4, GAL4; U, UAS.


E , F period of DD locomotor rhythm of flies when knocking down Nipped - A over - expressing not . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with not overexpression flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not Nipped - A knocked down .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The period of DD locomotor rhythm of flies when knocking down Nipped-A and over-expressing not. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


G period of DD locomotor rhythm of flies co - expressing Nipped - A RNAi not RNAi . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with not overexpression flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not Nipped - A knocked down .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 period of DD locomotor rhythm of flies co-expressing Nipped-A RNAi and not RNAi. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


( A ) siRNA - treated BMDMs were infected for 16 hours with S . Typhimurium ( orgA - ) LDH IL - 1β release were measured .

O O O O 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) siRNA-treated BMDMs were infected for 16 hours with S. Typhimurium (orgA-) and LDH and IL-1β release were measured.


( B ) ( LDH ) IL - 1β release evaluation in WT , Irgm2 - / - , GBPChr3 - / - Casp11 - / - BMDMs infected for 16 hours with different Gram - negative ( MOI 25 ) .

O O O O O O O O O O O O O O O O O O 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) Cell death (LDH) and IL-1β release evaluation in WT, Irgm2-/-, GBPChr3-/- and Casp11-/- BMDMs infected for 16 hours with different Gram-negative bacteria (MOI 25).


( C ) Western blot examination of processed caspase - 1 ( p20 ) gasdermin - D ( p30 ) in supernatants pro - caspase - 1 ( p45 ) , pro - gasdermin - D ( p55 ) GAPDH in lysates of WT Irgm2 - / - BMDMs infected for 16 h with different Gram - negative strains .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 examination of processed caspase-1 (p20) and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55) and GAPDH in cell lysates of WT and Irgm2-/- BMDMs infected for 16 h with different Gram-negative bacterial strains.


( D ) IL - 1β ( % LDH ) evaluation in immortalized WT , Irgm2 - / - , Casp11 - / - Casp11 - / - Irgm2 - / - ( referred as sgIrgm2 ) BMDMs after 16 hours of E . coli , S . Typhimurium orgA - OMV treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IL-1β and cell death (% LDH) evaluation in immortalized WT, Irgm2-/-, Casp11-/- and Casp11-/-Irgm2-/- (referred as sgIrgm2) BMDMs after 16 hours of E. coli, S. Typhimurium orgA- and OMV treatment.


( E ) ( % LDH ) evaluation in IFNγ - primed WT , Irgm2 - / - Casp11 - / - BMDMs 4 hours after electroporation or not with 1µg of E . coli LPS .

O O O O O O O O O O O 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

(E) Cell death (% LDH) evaluation in IFNγ-primed WT, Irgm2-/- and Casp11-/- BMDMs 4 hours after electroporation or not with 1µg of E. coli LPS.


( F - H ) Survival of WT , Casp11 - / - Irgm2 - / - mice primed with 100µg poly ( I : C ) for 6 hours injected ( i . p . ) with 5mg / k - 1 LPS or 5 25 µg of OMVs ( n = 6 per condition ) .

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

(F-H) Survival of WT, Casp11-/- and Irgm2-/- mice primed with 100µg poly(I:C) for 6 hours and injected (i.p.) with 5mg/k-1 LPS or 5 and 25 µg of OMVs (n=6 animals per condition).


( A ) Measure of LDH IL - 1β release in WT , GBPChr3 - / - Casp11 - / - BMDMs were Irgm2 was knocked down 16 hours after exposure to 2 , 5 µg / 2 . 105 of OMVs . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Measure of LDH and IL-1β release in WT, GBPChr3-/- and Casp11-/- BMDMs were Irgm2 was knocked down 16 hours after exposure to 2,5 µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( B ) ( LDH ) IL - 1β release evaluation in Irgm2 - silenced WT GBPChr3 - / - BMDMs infected for 16 hours with either S . Tm orgA - or F . novicida ( MOI 25 ) . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell death (LDH) and IL-1β release evaluation in Irgm2-silenced WT and GBPChr3-/- BMDMs infected for 16 hours with either S.Tm orgA- or F. novicida (MOI 25). Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( C ) Florescence microscopy associated quantifications of GBP - 2 ( green ) recruitments to intracellular S . Tm orgA - - mCherry ( MOI 10 , red ) in IFNγ - primed WT Irgm2 - / - BMDMs . Nucleus was stained with Hoechst ( blue ) . Confocal shown are from one experiment are representative of n = 3 independent experiments ; scale bars 5 µm . For quantifications , of GBP - associated was quantified ' n = ' refers to number of intracellular counted in each experiment ; quantifications from n = 3 independent experiments were then plotted expressed as mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Florescence microscopy and associated quantifications of GBP-2 (green) recruitments to intracellular S. Tm orgA--mCherry (MOI 10, red) in IFNγ-primed WT and Irgm2-/-BMDMs. Nucleus was stained with Hoechst (blue). Confocal images shown are from one experiment and are representative of n=3 independent experiments; scale bars 5 µm. For quantifications, the percentage of GBP-associated bacteria was quantified and 'n=' refers to the number of intracellular bacteria counted in each experiment; quantifications from n=3 independent experiments were then plotted and expressed as mean ± SEM.


( D ) Confocal microscopy associated quantifications of caspase - 11 - C254G - GFP ( green ) recruitment to S . Tm - mCherry ( orgA - , red ) in IFNγ - primed iWT , iIrgm2 - / - , iGBPChr3 - / - iGBPChr3 - / - / sgIrgm2 BMDMs after 8 hours of infection . Nucleus ( blue ) was stained with Hoescht ; scale bar 5µm . For quantifications , of positive for caspase - 11 - C254G - GFP was determined by combining from n = 3 independent experiments expressed as mean ± . * * * p ≤ 0 . 001 for indicated comparisons using t - with bonferroni correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red) in IFNγ-primed iWT, iIrgm2-/-, iGBPChr3-/- and iGBPChr3-/-/sgIrgm2 BMDMs after 8 hours of infection. Nucleus (blue) was stained with Hoescht; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction.


( A ) GFP - Trap coupled to mass spectrometry strategy used . volcano plot represents 3 independent combined experiments . Threshold selection of enriched ( red dots ) in Irgm2 - GFP fraction was set at 2 fold enrichment ( x axis ) p - value < 0 . 05 ( y axis ) . Blacks Grey dots indicate that did not reach a p - value < 0 . 05 using t - with bonferroni correction . Labelled represent top 6 enriched in Irgm2 fraction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-Trap coupled to mass spectrometry strategy used. The volcano plot represents 3 independent combined experiments. Threshold selection of enriched proteins (red dots) in Irgm2-GFP fraction was set at 2 fold enrichment (x axis) and p-value <0.05 (y axis). Blacks and Grey dots indicate proteins that did not reach a p-value <0.05 using t-test with bonferroni correction. Labelled proteins represent the top 6 enriched proteins in the Irgm2 fraction.


( B ) Green Florescent ( GFP ) - Trap of presence of Gate16 in Irgm2 - GFP - enriched fraction from lysates of IFNγ - primed iIrgm2 - / - BMDMs complemented with lentiviral constructs coding for a fusion of Irgm2 withGFP ( Irgm2 - GFP ) or GFP alone . Arrows show presence or not of Gate16 , Irgm2 - GFP GAPDH in Irgm2 - GFP - enriched fractions , flow - through total lysates . * means non - specific

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Green Florescent Protein (GFP)-Trap assay of the presence of Gate16 in Irgm2-GFP-enriched fraction from the lysates of IFNγ-primed iIrgm2-/- BMDMs complemented with lentiviral constructs coding for a fusion of Irgm2 withGFP (Irgm2-GFP) or GFP alone. Arrows show the presence or not of Gate16, Irgm2-GFP and GAPDH in the Irgm2-GFP-enriched fractions, flow-through and total cell lysates. * means non-specific band


( C ) LDH IL - 1β release from siRNA - treated WT BMDMs , then exposed to 2 . 5 µg / 2 . 105 of OMVs for 16 hours .

O O O O O O O O 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) LDH and IL-1β release from siRNA-treated WT BMDMs, and then exposed to 2.5 µg/2.105 cells of OMVs for 16 hours.


( D ) LDH IL - 1β release from WT , Irgm2 - / - Casp11 - / - BMDMs silenced for Gate16 treated for 16 hours with 2 , 5µg / 2 . 105 of OMVs . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

(D) LDH and IL-1β release from WT, Irgm2-/- and Casp11-/- BMDMs silenced for Gate16 and treated for 16 hours with 2,5µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( E ) Western blot examination of caspase - 11 processed caspase - 1 ( p20 ) gasdermin - D ( p30 ) in supernatants pro - caspase - 1 ( p45 ) , pro - gasdermin - D ( p55 ) , pro - caspase - 11 , Gate16 GAPDH in lysates of Gate16 - silenced WT , Irgm2 - / - GBPChr3 - / - BMDMs exposed to 2 , 5 . 105µg / 2 . 105 of OMVs for 16 hours . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot examination of caspase-11 and processed caspase-1 (p20)and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55), pro-caspase-11, Gate16 and GAPDH in cell lysates of Gate16-silenced WT, Irgm2-/- and GBPChr3-/- BMDMs exposed to 2,5.105µg/2.105 cells of OMVs for 16 hours. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( F ) Representative confocal microscopy associated quantifications of caspase - 11 - C254G - GFP ( green ) recruitment to S . Tm - mCherry ( orgA - , red , MOI 10 ) in IFNγ - primed iWT BMDMs silenced for Gate16 after 4 8 hours of infection . Nucleus ( blue ) was stained with Hoechst ; scale bar 5µm . For quantifications , of positive for caspase - 11 - C254G - GFP was determined by combining from n = 3 independent experiments expressed as mean ± . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 for indicated comparisons using t - with bonferroni correction . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red, MOI 10) in IFNγ-primed iWT BMDMs silenced for Gate16 after 4 and 8 hours of infection. Nucleus (blue) was stained with Hoechst; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( A ) LDH IL - 1B release from siRNA - treated human Monocyte - Derived Macrophages ( hMDMs ) infected with S . Typhimurium orgA - ( MOI25 ) for 16 hours . When specified , caspase - 4 / 5 inhibitor Z - LEVD ( 25µM ) or NLRP3 inhibitor MCC950 ( 10µM ) were added to experiments .

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(A) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs) infected with S. Typhimurium orgA- (MOI25) for 16 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.


( B ) LDH IL - 1B release from siRNA - treated human Monocyte - Derived Macrophages ( hMDMs ) , primed with IFNγ ( 10UI / mL ) PAM3CSK4 ( 100ng / mL ) then stimulated with Nigericin ( 20µM ) for 4 hours . When specified , caspase - 4 / 5 inhibitor Z - LEVD ( 25µM ) or NLRP3 inhibitor MCC950 ( 10µM ) were added to experiments .

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(B) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs), primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then stimulated with Nigericin (20µM) for 4 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.


( C , D ) LDH IL - 1B release from siRNA - treated WT or GBP1 / 2 / 5 - / - U937 Monocytic , primed with IFNγ ( 10UI / mL ) PAM3CSK4 ( 100ng / mL ) then infected with ( C ) with S . Typhimurium orgA - ( MOI25 ) for 10 hours or ( D ) electroporated with 1µg of E . coli LPS for 4 hours . Φ indicates that no LPS electroporation was performed .

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(C, D) LDH and IL-1B release from siRNA-treated WT or GBP1/2/5-/- U937 Monocytic cell line, primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then infected with (C) with S. Typhimurium orgA- (MOI25) for 10 hours or (D) electroporated with 1µg of E. coli LPS for 4 hours. Φ indicates that no LPS electroporation was performed.


B of differentiation ( left ) proliferation ( right ) in intestine after CDK8 deletion as in ( A ) . Olfm4 , Lysozyme , PAS Dclk1 was used to reveal , respectively , , Paneth , goblet tuft . β - Catenin allows detection of cancer ( vs localisation ) . proliferation was assessed by PCNA , Ki - 67 BrdU ( 2h ) . Scatter plots represent of stained by each antibody ( relative to occupied by hematoxylin ) . For Paneth , BrdU , PCNA Ki67 , only crypts were analysed . For goblet , crypts villi were analysed . For Tuft , Dclk1 - positive were counted in 50 villi . Colour code depicts small intestine ( green ) , proximal colon ( blue ) , distant colon ( red ) . Mean ± is shown . P - value of unpaired two - t - is indicated ( ns , not significant ; p > 0 . 05 ) . Scale bars , 25μm ( Olfm4 , Lysozyme , Dclk1 β - Catenin ) 50μm ( PAS , BrdU , Ki - 67 PCNA ) . ( n = 9 biological replicates )

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B Analysis of cell differentiation (left) and proliferation (right) in the intestine after CDK8 deletion as in (A). Olfm4, Lysozyme, PAS and Dclk1 staining was used to reveal, respectively, stem, Paneth, goblet and tuft cells. β-Catenin staining allows detection of cancer cells (cytoplasmic vs nuclear localisation). Cell proliferation was assessed by PCNA, Ki-67 and BrdU (2h pulse) staining. Scatter plots represent the percentage of the area stained by each antibody (relative to the area occupied by hematoxylin). For Paneth cells, BrdU, PCNA and Ki67, only crypts were analysed. For goblet cells, crypts and villi were analysed. For Tuft cell quantification, Dclk1-positive cells were counted in 50 villi. Colour code depicts small intestine (green), proximal colon (blue), and distant colon (red). Mean ± SEM is shown. P-value of unpaired two-tailed t-test is indicated (ns, not significant; p > 0.05). Scale bars, 25μm (Olfm4, Lysozyme, Dclk1 and β-Catenin) and 50μm (PAS, BrdU, Ki-67 and PCNA). (n=9 biological replicates)


C of mouse colon after AOM / DSS treatment . of number of neoplastic lesions ( n = 10 for Cdk8 + / + , n = 7 for Cdk8 - / - mice ) . P - value of unpaired t - is indicated : ns , not significant ( p > 0 . 05 ) . Mean ± SD is shown .

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C Analysis of mouse colon after AOM/DSS treatment. Quantification of the number of neoplastic lesions (n = 10 for Cdk8+/+, and n = 7 for Cdk8 -/- mice). P-value of unpaired t-test is indicated: ns, not significant (p > 0.05). Mean ± SD is shown.


D of of colon surface occupied by tumours . Intestine samples were stained for β - Catenin , tumour regions with β - Catenin localisation were quantified using NDP . view software . Two - p - value of unpaired t - is indicated ; ns , not significant ( p > 0 . 05 ) . Mean ± SD is shown . ( n = 6 biological replicates )

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D Quantification of the percentage of the colon surface occupied by tumours. Intestine samples were stained for β-Catenin, and tumour regions with nuclear β-Catenin localisation were quantified using NDP.view software. Two-tailed p-value of unpaired t-test is indicated; ns, not significant (p > 0.05). Mean ± SD is shown. (n=6 biological replicates)


E Example of with β - Catenin of tumour - free regions with β - catenin localisation ( left ) tumour regions with β - catenin localisation ( right ) . Scale bars , 50μm .

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E Example of IHC with β-Catenin staining of tumour-free regions with membrane β-catenin localisation (left) and tumour regions with nuclear β-catenin localisation (right). Scale bars, 50μm.


A Genotyping confirms loss of Cdk8 exon 2 in Cdk8 - / - Cdk8 - / - / Cdk19 - / - organoids after 7 days of OH - tamoxifen treatment .

A Genotyping confirms the loss of Cdk8 exon 2 in Cdk8 -/- and Cdk8 -/-/Cdk19 -/- organoids after 7 days of OH-tamoxifen treatment.


B WB of organoid samples after 7 days of OH - tamoxifen treatment ; β - actin was used as loading control .

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

B WB of organoid samples after 7 days of OH-tamoxifen treatment; β-actin was used as loading control.


C Phase contrast of organoids before after 6 days of OH - tamoxifen treatment . Scale bars , 150μm . D of organoid at day 6 , shown in ( C ) ( mean ± are shown ) . P - value , one - way ANOVA ( Bonferroni ) : ( * ) p ≤ 0 . 05 , ( * * * ) p ≤ 0 . 001 ; ns , not significant ( p > 0 . 05 ) . ( n = 47 biological replicates )

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C Phase contrast images of organoids before and after 6 days of OH-tamoxifen treatment. Scale bars, 150μm. D Quantification of organoid size at day 6, shown in (C) (mean ± SEM are shown). P-value, one-way ANOVA (Bonferroni test): (*) p ≤ 0.05, (***) p ≤ 0.001; ns, not significant (p > 0.05). (n=47 biological replicates)


E of organoids ( day 7 of OH - tamoxifen treatment ) with Ki - 67 antibody . Scale bars , 100μm . F of Ki - 67 positive ( % of total of organoids ; mean ± ) in four different genotypes presented in ( E ) . Areas with positive Ki - 67 signal were detected quantified using QuPath ImageJ programs . Four technical replicates from 2 biological replicates of each genotype were analysed . Adjusted p - values of ordinary one - way ANOVA ( Bonferroni ) are indicated : ( * * ) p - values ≤ 0 . 01 ; ( * ) p - values ≤ 0 . 05 ; ns , not significant ( p > 0 . 05 ) .

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E IHC staining of organoids (day 7 of OH-tamoxifen treatment) with Ki-67 antibody. Scale bars, 100μm. F Quantification of Ki-67 positive area (% of the total area of the organoids; mean ± SEM) in the four different genotypes presented in (E). Areas with positive Ki-67 signal were detected and quantified using QuPath and ImageJ programs. Four technical replicates from 2 biological replicates of each genotype were analysed. Adjusted p-values of ordinary one-way ANOVA (Bonferroni test) are indicated: (**) p-values ≤ 0.01; (*) p-values ≤ 0.05; ns, not significant (p > 0.05).


C Left , of WT Cdk8 - / - / Cdk19 - / - organoids with Olfm4 ( ) , Lysozyme ( Paneth ) , Muc2 ( goblet ) , Dclk1 ( tuft ) antibodies . Scale bars , 50μm ( Olfm4 , Muc2 , Dclk1 ) 25μm ( Lysozyme ) . Right , of Olfm4 , Lysozyme Muc2 positive ( % of total organoid as indicated by hematoxylin ; mean ± ) , or number of Dclk1 positive per organoid . P - value , unpaired two - t - : ( * * * ) p - value ≤ 0 . 001 ; ns , not significant ( p > 0 . 05 ) . 20 different organoids from 2 biological replicates of each genotype were used for .

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C Left, IHC staining of WT and Cdk8 -/-/Cdk19 -/- organoids with Olfm4 (stem cells), Lysozyme (Paneth cells), Muc2 (goblet cells), and Dclk1 (tuft cells) antibodies. Scale bars, 50μm (Olfm4, Muc2, Dclk1) and 25μm (Lysozyme). Right, quantification of Olfm4, Lysozyme and Muc2 positive area (% of the total organoid area as indicated by hematoxylin staining; mean ± SEM), or the number of Dclk1 positive cells per organoid. P-value, unpaired two-tailed t-test: (***) p-value ≤ 0.001; ns, not significant (p > 0.05). 20 different organoids from 2 biological replicates of each genotype were used for the analysis.


D WB of indicated extracted from Wt Cdk8 - / - / Cdk19 - / - organoids . Included are CFTR positive negative control samples obtained from Cystic Fibrosis Foundation ( CFF ) . Amidoblack was used as loading control .

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D WB of indicated proteins extracted from Wt and Cdk8 -/-/Cdk19 -/- organoids. Included are CFTR positive and negative control cell line samples obtained from the Cystic Fibrosis Foundation (CFF). Amidoblack was used as loading control.


A Histological PAS of organoids treated for 7 days with OH - tamoxifen . Scale bar , 50μm . B of PAS signal ( % of total organoid ; mean ± SD are shown ) in four different genotypes presented in ( A ) . Areas containing positive PAS were detected quantified using QuPath ImageJ programs . Adjusted p - values of ordinary one - way Anova followed by Tukey ' s multiple comparison are indicated : ( * * * ) p - value ≤ 0 . 001 ; ns : not significant ( p > 0 . 05 ) . Four technical replicates from 2 biological replicates of each genotype were analysed .

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A Histological PAS staining of organoids treated for 7 days with OH-tamoxifen. Scale bar, 50μm. B Quantification of PAS signal (% of total organoid area; mean ± SD are shown) in the four different genotypes presented in (A). Areas containing positive PAS staining were detected and quantified using QuPath and ImageJ programs. Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05). Four technical replicates from 2 biological replicates of each genotype were analysed.


C Representative phase contrast of organoids at indicated time points after 7 days of OH - tamoxifen treatment are shown . Scale bar , 100 μm . D of time needed for mucus release ( observed as a in center of organoid ; mean ± SD are shown ) . Adjusted p - values of ordinary one - way Anova followed by Tukey ' s multiple comparison are indicated : ( * * * ) p - value ≤ 0 . 001 ; ns : not significant ( p > 0 . 05 ) ; ( n = 17 for Wt , 11 for Cdk8 - / - , 18 for Cdk19 - / - , 12 for Cdk8 - / - / Cdk19 - / - , biological replicates ) .

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C Representative phase contrast images of organoids at the indicated time points after 7 days of OH-tamoxifen treatment are shown. Scale bar, 100 μm. D Quantification of the time needed for mucus release (observed as a dark staining in the center of the organoid; mean ± SD are shown). Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05); (n= 17 for Wt, 11 for Cdk8 -/-, 18 for Cdk19 -/-, and 12 for Cdk8 -/-/ Cdk19 -/- , all biological replicates).


E confocal microscopy of Calcein green - labeled WT Cdk8 - / - / Cdk19 - / - organoids treated with forskolin . Scale bars , 100 μm . F of forskolin - induced swelling in WT organoids treated for 1hour or 24 hours with 0 . 1μM , 1μM or 10μM Senexin B ( SenB ) , as indicated , or double KO organoids ; DMSO vehicle was used as control . surface of organoids at different time points relative to at t = 0 ( 100 % ) was measured ( mean ± SD , n = 8 biological replicates ) . Linear regression lines are shown .

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E Fluorescence confocal microscopy images of Calcein green-labeled WT and Cdk8-/-/Cdk19-/- organoids treated with forskolin. Scale bars, 100 μm. F Quantification of forskolin-induced swelling in WT organoids treated for 1hour or 24 hours with 0.1μM, 1μM or 10μM Senexin B (SenB), as indicated, or double KO organoids; DMSO vehicle was used as control. The surface area of individual organoids at different time points relative to the area at t = 0 (100%) was measured (mean ± SD, n=8 biological replicates). Linear regression lines are shown.


H WB of indicated extracted from Senexin B - treated ( 10μM , 2h 24h ) organoids ( left ) , WT Cdk8 - / - / Cdk19 - / - organoids ( right ) . Amidoblack was used as loading control .

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H WB of indicated proteins extracted from Senexin B-treated (10μM, 2h and 24h) organoids (left), and WT and Cdk8 -/-/ Cdk19 -/- organoids (right). Amidoblack was used as loading control.


A WB of indicated extracted from intestinal epithelium of control mice or mice treated with Senexin 631 ( Snx 631 ) for 4 or 11 days .

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A WB of indicated proteins extracted from intestinal epithelium of control mice or mice treated with Senexin 631 (Snx 631) for 4 or 11 days.


B Left , immunohistochemical PAS hematoxylin of mouse small intestines collected at 4 11 days of Senexin 631 ( Snx 631 ) treatment ; scale bars 50μm . Right , of PAS - hematoxilin ( Hema ) - positive ( mean ± ) , using QuPath ImageJ software . Adjusted p - values of unpaired t - are shown : ( * ) p < 0 . 05 ; ns , not significant ( p > 0 . 05 ) . 8 mice ( 4 treated with Senexin 631 4 control ) were used in this .

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B Left, immunohistochemical PAS and hematoxylin staining of mouse small intestines collected at 4 and 11 days of Senexin 631 (Snx 631) treatment; scale bars 50μm. Right, quantification of PAS- and hematoxilin (Hema)-positive area (mean ± SEM), using QuPath and ImageJ software. Adjusted p-values of unpaired t-test are shown: (*) p < 0.05; ns, not significant (p > 0.05). 8 mice (4 treated with Senexin 631 and and 4 control) were used in this quantification.


Changes with time in amounts of SH - EP , α - amylase , VmPE - 1 in cotyledons of V . mungo seedlings ( Attached cotyledons ) or axis - removed cotyledons of V . mungo seeds ( Detached cotyledons ) . Attached or detached cotyledons were prepared as described in Materials methods . 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris - Cl ( pH 7 . 4 ) , homogenates were centrifuged at 15 , 000 g for 10 min . Supernatants ( 10 μl each ) were analyzed by SDS - PAGE immunoblotting with anti - SH - EP , α - amylase , or VmPE - 1 antibody .

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Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V. mungo seedlings (Attached cotyledons) or embryo axis-removed cotyledons of V. mungo seeds (Detached cotyledons). Attached or detached cotyledons were prepared as described in Materials and methods. 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris-Cl (pH 7.4), and the homogenates were centrifuged at 15,000 g for 10 min. Supernatants (10 μl each) were analyzed by SDS-PAGE immunoblotting with anti-SH-EP, α-amylase, or VmPE-1 antibody.


Subcellular distribution of α - amylase in V . mungo cotyledon . Microsome PSV fractions were prepared from cotyledons of day 3 seedlings as described in Materials methods . Extracts from dry seeds day 3 cotyledons , microsomes , PSV fractions ( 20 μg each ) were separated by SDS - PAGE . in gel were analyzed by Coomassie Brilliant blue ( A ) , or by immunoblotting with anti - SH - EP antibody ( B ) or anti - α - amylase antibody ( C ) . Bracket indicates polypeptides derived from storage globulins . Arrowhead indicates α - amylase .

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Subcellular distribution of α-amylase in V. mungo cotyledon cells. Microsome and PSV fractions were prepared from cotyledons of day 3 seedlings as described in Materials and methods. Extracts from dry seeds and day 3 cotyledons, microsomes, and PSV fractions (20 μg protein each) were separated by SDS-PAGE. Proteins in the gel were analyzed by Coomassie Brilliant blue staining (A), or by immunoblotting with anti-SH-EP antibody (B) or anti- α-amylase antibody (C). Bracket indicates the polypeptides derived from storage globulins. Arrowhead indicates α-amylase.


Electron showing immunogold localization of α - amylase in attached cotyledons ( A - E ) , detached cotyledons ( F ) , or control for immunogold labeling ( G ) . ( A ) Anti - α - amylase antibody immunogold - stained PSV , but not SG . ( B C ) Golgi PSVs were both immunogold - labeled with anti - α - amylase antibody . ( D ) Immunogold localization of α - amylase ( 15 - nm particles ) SH - EP ( 10 - nm particles ) . α - Amylase SH - EP were localized in PSVs KVs , respectively . ( E ) Gold particles from anti - α - amylase antibody were detected in LVs as well as PSVs . ( F ) PSVs in detached cotyledons were immunogold labeled with anti - α - amylase antibody . ( G ) Immunogold of detached cotyledon without first antibody ( anti - α - amylase antibody ) . No gold particles were observed in . CW , wall ; G , Golgi ; KV , KDEL - cysteine proteinase - accumulating vesicle ; LV , lytic vacuole ; Mt , mitochondrion ; PSV , storage vacuole ; SG , starch granule . Bars , 200 nm .

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Electron micrographs showing the immunogold localization of α-amylase in attached cotyledons cells (A-E), detached cotyledons cells (F), or control for immunogold labeling (G). (A) Anti-α-amylase antibody immunogold-stained PSV, but not SG. (B and C) Golgi complex and PSVs were both immunogold-labeled with anti- α-amylase antibody. (D) Immunogold localization of α-amylase (15-nm particles) and SH-EP (10-nm particles). α-Amylase and SH-EP were localized in PSVs and KVs, respectively. (E) Gold particles from anti-α-amylase antibody were detected in LVs as well as PSVs. (F) PSVs in detached cotyledons were immunogold labeled with anti- α-amylase antibody. (G) Immunogold staining of detached cotyledon cells without first antibody (anti-α-amylase antibody). No gold particles were observed in the cell. CW, cell wall; G, Golgi complex; KV, KDEL-tailed cysteine proteinase-accumulating vesicle; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.


Electron showing ultrastructures of cotyledon of germinatedV . mungo seeds ( A - G ) , immunogold localization of α - amylase in cotyledon ( H ) , ultrastructure of of detached cotyledons ( I ) . Toluidine blue ( TB ) of sections from cotyledons of day 3 V . mungo seedlings . Conversion from TB - stained ( region I ) to TB - stainless ( region III ) was accompanied with that of PSV to LV . ( B ) SGs PSVs in TB - stained ( A , region I ) . PSV was filled with storage . Arrowheads indicate border between SGs cytoplasm . ( C ) SGs PSVs in cotyledons at region II in A . Electron density of PSVs became low . SGs were surrounded with membranous structure ( arrows ) . LED areas were found between SGs cytoplasm . ( D ) SG LV in TB - stainless ( region III in A ) . PSV was converted to LV in . areas around SG with LED were enlarged . ( E ) Ultrastructure of TB - stainless . SGs wrapped with a LED contacted with LVs ( arrowheads ) . Vesicles with similar density to LVs were observed . ( F ) LED membranes around SGs fused with LV membranes ( arrow ) . ( G ) SGs were observed in LVs . shape of SGs were largely different from those in B - E . ( H ) An Immunogold image representing degradation of SGs by α - amylase localized in LVs . Gold particles from anti - α - amylase antibody were densely detected in peripheral region of SGs , which is inserted into inside of LV . ( I ) PSVs were not converted to LVs , SGs were not taken up into PSVs in of detached cotyledons . Neither low density areas around SGs nor vesicles with similar density to LV was observed in . LV , lytic vacuole ; Mt , mitochondrion ; PSV , storage vacuole ; SG , starch granule . Bars : ( A ) 50 μm ; ( E G ) 2 μm ; ( D , F , I ) 1 μm ; ( B , C , H ) 200 nm .

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

Electron micrographs showing ultrastructures of cotyledon cells of germinatedV. mungo seeds (A-G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B-E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti-α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.


LysoTracker red of partially broken cotyledon , SGs , or cotyledon sections . Cotyledon , SGs , sections were prepared from day 3 attached or detached cotyledons stained with LysoTracker red as described in Materials methods . ( A ) Differential interface - contrast image of a broken . ( B ) image of A . ( C ) Differential interface - contrast image of a broken . ( D ) image of C . ( E ) Differential interface - contrast image of of a section from attached cotyledons . ( F ) image of E . ( G ) Magnified image of E . ( H ) Magnified image of F . ( I ) Differential interface - contrast image of an SG . ( J ) image of I . ( K ) Differential interface - contrast image of an SG . ( L ) image of K . ( M ) Differential interface - contrast image of of a section from detached cotyledons . ( N ) image of M . ( O ) Magnified image of M . ( P ) Magnified image of O . Bars : ( A - D , G - L , O , P ) 20 μm ; ( E , F , M , N ) 50 μm .

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

LysoTracker red staining of partially broken cotyledon cells, SGs, or cotyledon sections. Cotyledon cells, SGs, and sections were prepared from day 3 attached or detached cotyledons and stained with LysoTracker red as described in Materials and methods. (A) Differential interface-contrast image of a broken cell. (B) Fluorescent image of A. (C) Differential interface-contrast image of a broken cell. (D) Fluorescent image of C. (E) Differential interface-contrast image of cells of a section from attached cotyledons. (F) Fluorescent image of E. (G) Magnified image of E. (H) Magnified image of F. (I) Differential interface-contrast image of an SG. (J) Fluorescent image of I. (K) Differential interface-contrast image of an SG. (L) Fluorescent image of K. (M) Differential interface-contrast image of cells of a section from detached cotyledons. (N) Fluorescent image of M. (O) Magnified image of M. (P) Magnified image of O. Bars: (A-D, G-L, O, and P) 20 μm; (E, F, M, and N) 50 μm.


Electron showing autophagosome - mediated autophagy in attached ( A - C ) detached ( D E ) cotyledon . ( A ) Two autophagosomes containing cytoplasm were observed . Mitochondria cytoplasm appeared to be enclosed with an ER - like double . ( B ) An autophagosome containing mitochondria cytoplasm was fused with LV ( arrowheads ) . ( C ) Autophagic containing mitochondria was found in LV . ( D ) An autophagosome containing mitochondria cytoplasm was observed . PSV was not converted into LV . ( E ) Autophagic was observed in PSV of detached cotyledon . AB , autophagic ; AP , autophagosome ; ER , endoplasmic reticulum ; LV , lytic vacuole ; Mt , mitochondrion ; PSV , storage vacuole ; SG , starch granule . Bars , 200 nm .

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

Electron micrographs showing autophagosome-mediated autophagy in attached (A-C) and detached (D and E) cotyledon cells. (A) Two autophagosomes containing cytoplasm were observed. Mitochondria and the cytoplasm appeared to be enclosed with an ER-like double membrane. (B) An autophagosome containing mitochondria and cytoplasm was fused with LV (arrowheads). (C) Autophagic body containing mitochondria was found in LV. (D) An autophagosome containing mitochondria and cytoplasm was observed. The PSV was not converted into the LV. (E) Autophagic body was observed in the PSV of detached cotyledon cells. AB, autophagic body; AP, autophagosome; ER, endoplasmic reticulum; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.


E representative of IF - of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) DAPI ( blue ) . Scale bar , 200 μm . F of numbers of Iba - 1 + on meningeal regions . Mock n = 5 ; ZIKV n = 7 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - unpaired t - . data are presented as mean ± .

E The representative images of IF-IHC staining of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 200 μm. F Quantification of numbers of Iba-1+ cells on the meningeal regions. Mock n=5; ZIKV n=7 embryos. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM.


G representative of IF - of choroid plexus of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) DAPI ( blue ) . Scale bar , 50 μm . H of total number of Iba - 1 + per ChP . I of Iba - 1 + on surface of ChP . J of round - shaped Iba - 1 + . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - unpaired t - . data are presented as mean ± . Mock n = 5 ; ZIKV n = 6 biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 50 μm. H Quantification of total number of Iba-1+ cells per ChP area. I Percentage of Iba-1+ cells on the surface of ChP. J Percentage of round-shaped Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM. Mock n=5; ZIKV n=6 biological replicates


K representative of IF - of choroid plexus of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) , CD68 ( red ) DAPI ( blue ) . Scale bar , 50 μm . L of CD68 + ; Iba - 1 + out of total Iba - 1 + . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - unpaired t - . data are presented as mean ± .

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

K The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green), CD68 (red) and DAPI (blue). Scale bar, 50 μm. L Percentage of CD68+; Iba-1+ cells out of total Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM.


B Representative tracks in sociability . C of time subject mice spent in each chamber in sociability session of three - chamber . Mock ( n = 11 ) stranger 1 ( S1 ) chamber vs . empty ( E ) chamber ; ZIKV ( n = 16 ) stranger 1 chamber vs . empty chamber ; one - way ANOVA with Bonferroni post hoc . D of time spent investigating stranger mouse empty cage during sociability session . Mock ( n = 11 ) sniffing time at stranger 1 mouse vs . empty cup , ZIKV ( n = 16 ) sniffing time at stranger 1 mouse vs . empty cup . E Preference indices in sociability . Mock ( n = 11 ) vs . ZIKV ( n = 16 ) . F Representative tracks in social memory . G of time subject mice spent in each in social memory session . Mock ( n = 11 ) stranger 1 ( S1 ) chamber vs . stranger 2 ( S2 ) chamber , p < 0 . 0001 ; ZIKV ( n = 16 ) stranger 1 chamber vs . stranger 2 chamber ; one - way ANOVA with Bonferroni post hoc . H of time spent investigating familiar mouse novel stranger mouse during social novelty session . Mock ( n = 11 ) sniffing time at stranger 1 vs . stranger 2 mouse ; ZIKV ( n = 16 ) sniffing time at stranger 1 vs . stranger 2 mouse . I Preference indices in social novelty session of three - chamber . Mock ( n = 11 ) vs . ZIKV ( n = 16 ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - unpaired t - unless described otherwise ; n represent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative animal tracks in the sociability test. C Percentage of time the subject mice spent in each chamber in the sociability session of three-chamber test. Mock (n=11) stranger 1 (S1) chamber vs. empty (E) chamber; ZIKV (n=16) stranger 1 chamber vs. empty chamber; one-way ANOVA with Bonferroni post hoc test. D Quantification of time spent investigating the stranger mouse and empty cage during sociability session. Mock (n=11) sniffing time at stranger 1 mouse vs. empty cup, ZIKV (n=16) sniffing time at stranger 1 mouse vs. empty cup. E Preference indices in sociability. Mock (n=11) vs. ZIKV (n=16). F Representative animal tracks in the social memory test. G Percentage of time the subject mice spent in each in the social memory session. Mock (n=11) stranger 1 (S1) chamber vs. stranger 2 (S2) chamber, p< 0.0001; ZIKV (n=16) stranger 1 chamber vs. stranger 2 chamber; one-way ANOVA with Bonferroni post hoc test. H Quantification of time spent investigating the familiar mouse and novel stranger mouse during social novelty session. Mock (n=11) sniffing time at stranger 1 vs. stranger 2 mouse; ZIKV (n=16) sniffing time at stranger 1 vs. stranger 2 mouse. I Preference indices in the social novelty session of the three-chamber test. Mock (n=11) vs. ZIKV (n=16). Data information: All data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired t-test unless described otherwise; n represent biological replicates.


B Quantitation of sEPSC frequency mPFC from pyramidal neurons in mPFC . C Quantitation of sEPSC amplitude of mPFC pyramidal neurons . Mock , n = 14 neurons from 4 mice ; ZIKV , n = 15 neurons from 5 mice in B C . Data information : Data were collected from PL IL regions of mPFC , were presented as mean ± . * p < 0 . 05 , * * p < 0 . 01 . Two - unpaired t - unless described otherwise .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantitation of sEPSC frequency mPFC from pyramidal neurons in mPFC. C Quantitation of sEPSC amplitude of mPFC pyramidal neurons. Mock, n=14 neurons from 4 mice; ZIKV, n= 15 neurons from 5 mice in B and C. Data information: Data were collected from PL and IL regions of mPFC, and were presented as mean ± SEM. *p<0.05, **p<0.01. Two-tailed unpaired t-test unless described otherwise.


E Quantifications of sIPSC frequency from pyramidal neurons in mPFC . F Quantifications of sIPSC amplitude of mPFC pyramidal neurons . Mock , n = 14 neurons from 5 mice ; ZIKV , n = 18 neurons from 6 mice in E F . Data information : Data were collected from PL IL regions of mPFC , were presented as mean ± . * p < 0 . 05 , * * p < 0 . 01 . Two - unpaired t - unless described otherwise .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantifications of sIPSC frequency from pyramidal neurons in mPFC. F Quantifications of sIPSC amplitude of mPFC pyramidal neurons. Mock, n=14 neurons from 5 mice; ZIKV, n= 18 neurons from 6 mice in E and F. Data information: Data were collected from PL and IL regions of mPFC, and were presented as mean ± SEM. *p<0.05, **p<0.01. Two-tailed unpaired t-test unless described otherwise.


A Representative of PV + , SST + CAL + in prelimbic ( PL , upper panel ) infralimbic ( IL , lower panel ) cortex . Scale bars : 200 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

A Representative images of PV+, SST+ and CAL+ cells in the prelimbic (PL, upper panel) and infralimbic (IL, lower panel) cortex. Scale bars: 200 μm.


H of mPFC stained with antibodies against vGLUT1 . Scale bars , 5 μm . I of vGLUT1 + numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 . Two - unpaired t - unless described otherwise .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 IHC staining of mPFC stained with antibodies against vGLUT1. Scale bars, 5 μm. I Quantification of vGLUT1+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice. Data information: All data are presented as mean ± SEM. *P<0.05, **P<0.01. Two-tailed unpaired t-test unless described otherwise.


J of mPFC stained with antibodies against vGAT . Scale bars , 5 μm . K of vGAT + numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 . Two - unpaired t - unless described otherwise .

J IHC staining of mPFC stained with antibodies against vGAT. Scale bars, 5 μm. K Quantification of vGAT+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice. Data information: All data are presented as mean ± SEM. *P<0.05, **P<0.01. Two-tailed unpaired t-test unless described otherwise.


B Representative confocal of vHIP stained with mCherry ( red ) c - Fos ( green ) . C of mCherry c - Fos double positive out of total mCherry - positive in vHIP CA1 regions . Scale bar , 50 μm . Mock ( n = 5 mice ) vs . ZIKV ( n = 5 mice ) , p = 0 . 0013 . Data information : Data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - unless described otherwise .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative confocal images of the vHIP stained with mCherry (red) and c-Fos (green). C Quantification of mCherry and c-Fos double positive cells out of total mCherry-positive cells in the vHIP CA1 regions. Scale bar, 50 μm. Mock (n= 5 mice) vs. ZIKV (n= 5 mice), p=0.0013. Data information: Data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


F Representative EPSC traces of pyramidal neurons in mPFC . G of EPSC amplitude of pyramidal neurons in mPFC ( PL IL ) . Mock ( n = 17 neurons from 5 mice ) vs . ZIKV ( n = 19 neurons from 5 mice ) , p = 0 . 0122 . Data information : Data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - unless described otherwise .

F Representative EPSC traces of the pyramidal neurons in the mPFC. G Quantification of EPSC amplitude of the pyramidal neurons in the mPFC (PL and IL). Mock (n=17 neurons from 5 mice) vs. ZIKV (n=19 neurons from 5 mice), p=0.0122. Data information: Data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


B Representative examples of GFP - labeled presynaptic boutons . Scale bars : 2 . 5 μm . C of presynaptic bouton density weighted in mPFC ( PL IL ) . Mock : n = 57 axons from 6 mice ; ZIKV : n = 63 axons from 7 mice . D of presynaptic bouton weighted in mPFC ( PL IL ) . Mock : n = 2974 boutons from 6 mice ; ZIKV : n = 3486 boutons from 7 mice . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - unless described otherwise .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative examples of GFP-labeled presynaptic boutons. Scale bars: 2.5 μm. C Quantification of presynaptic bouton density and weighted size in the mPFC (PL and IL). Mock: n= 57 axons from 6 mice; ZIKV: n= 63 axons from 7 mice. D Quantification of presynaptic bouton weighted size in the mPFC (PL and IL). Mock: n= 2974 boutons from 6 mice; ZIKV: n= 3486 boutons from 7 mice. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


H Comparisons of percentages of vHIP - innervated mPFC ( PL IL ) neuron subtypes . Mock , n = 4 mice ; ZIKV , n = 7 mice . P = 0 . 5343 in CaMKIIa , 0 . 0117 in PV , 0 . 7491 in CAL , 0 . 4484 in SST , 0 . 1519 in other uncharacterized neuron subtypes . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - unless described otherwise .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Comparisons of percentages of vHIP-innervated mPFC (PL and IL) neuron subtypes. Mock, n=4 mice; ZIKV, n=7 mice. P= 0.5343 in CaMKIIa, 0.0117 in PV, 0.7491 in CAL, 0.4484 in SST, 0.1519 in other uncharacterized neuron subtypes. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


J Representative confocal of CTB488 - labeled tdTomato - positive neurons in vHIP . arrowheads indicate CTB488 single positive neurons . Scale bars : 200 μm . K of of tdTomato ; CTB488 double - positive out of total CTB488 - positive . Mock , n = 7 mice ; ZIKV , n = 7 mice . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - unless described otherwise .

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

J Representative confocal images of CTB488-labeled tdTomato-positive neurons in the vHIP. White arrowheads indicate CTB488 single positive neurons. Scale bars: 200 μm. K Quantification of the percentage of tdTomato;CTB488 double-positive cells out of total CTB488-positive cells. Mock, n= 7 mice; ZIKV, n=7 mice. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


C Representative tracks in three - chamber . D Discrimination indices of social memory session of three - chamber . mCherry mock , n = 8 mice ; mCherry ZIKV , n = 10 mice ; hM4Di ZIKV n = 10 mice , mCherry mock vs . mCherry ZIKV p = 0 . 0041 ; mCherry ZIKV vs . hM4Di ZIKV p = 0 . 0115 ; mCherry mock vs . hM4Di ZIKV p = 0 . 7076 . Data information : data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 animal tracks in three-chamber test. D Discrimination indices of the social memory session of three-chamber test. mCherry mock, n=8 mice; mCherry ZIKV, n=10 mice; hM4Di ZIKV n=10 mice, mCherry mock vs. mCherry ZIKV p=0.0041; mCherry ZIKV vs. hM4Di ZIKV p=0.0115; and mCherry mock vs. hM4Di ZIKV p=0.7076. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.


E Representative confocal of mCherry ( red ) c - Fos ( green ) double - labeled in vHIP CA1 regions . Scale bars , 50 μm . F . of of c - fos ; mCherry double positive out of total mCherry . mCherry mock , n = 6 mice ; mCherry ZIKV , n = 4 mice ; hM4Di ZIKV n = 6 mice . Data information : data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative confocal images of mCherry (red) and c-Fos (green) double-labeled cells in the vHIP CA1 regions. Scale bars, 50 μm. F. Quantification of the percentage of c-fos;mCherry double positive cells out of total mCherry cells. mCherry mock, n=6 mice; mCherry ZIKV, n=4 mice; hM4Di ZIKV n=6 mice. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.


Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia . Select compounds were injected 24 hours before on day of LPS administration , using routes doses specified in methods . C57bl / 6 mice were injected with a high - lethality dose of E . coli LPS ( 38 - 40 μg / g ) followed by a subcutaneous injection of sterile saline . Significant differences in between drug vehicle - treated pre - post - pubertal mice are labeled with * * * * ( p < 0 . 0001 ) , * * * ( p < 0 . 001 , * * ( p < 0 . 01 ) , or * ( p < 0 . 05 ) . survival was compared using a log rank Mantel Cox

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

Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia. Select compounds were injected 24 hours before and on the day of LPS administration, using routes and doses specified in the methods. C57bl/6 female mice were injected with a high-lethality dose of E. coli LPS (38-40 μg/g) followed by a subcutaneous injection of sterile saline. Significant differences in concentration between drug and vehicle-treated pre- and post-pubertal mice are labeled with **** (p<0.0001), *** (p<0.001, ** (p<0.01), or * (p<0.05). Percent survival was compared using a log rank Mantel Cox test


D ) In 6 months old p32cKO heart , p62 multi - ubiquitin patterns were co - localized . Scale bar , 5 µm . E ) In 6 months old p32cKO heart , patterns of p62 mitochondrial marker , pyruvate dehydrogenase ( PDH ) , were co - localized . Scale bar , 5 µm .

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

D) In the 6 months old p32cKO heart, p62 and multi-ubiquitin staining patterns were co-localized. Scale bar, 5 µm. E) In the 6 months old p32cKO heart, the staining patterns of p62 and the mitochondrial marker protein, pyruvate dehydrogenase (PDH), were co-localized. Scale bar, 5 µm.