Figure 2A . InlK amino acid sequence . The signal sequence is underlined and the different regions of leucine rich repeats ( LRRs ) are outlined . The consensus pentapeptide LPXTG at the C - terminal end is boxed . B . Detection by immunofluorescence microscopy of InlK over - expressing in L . monocytogenes EGD - e ( WT ) , ΔinlK , WT + pADc - inlK , ΔinlK + pPRT - inlK and the ΔsrtA mutant over - expressing inlK ( ΔsrtA + pPRT - inlK ) grown in BHI medium using the rabbit polyclonal anti - InlK antibody . InlK was detected at the surface of InlK over - expressing bacteria ( WT + pADc - inlK and ΔinlK + pPRT - inlK ) , whereas it was undetectable at the surface WT bacteria or at the surface of the ΔstrA mutant over - expressing inlK . C . Detection of InlK by Western blot on total lysates of L . monocytogenes EGD - e ( WT ) , ΔinlK and ΔinlK + pPRT - inlK grown in BHI using the rabbit polyclonal anti - InlK antibody . Decreased concentrations of recombinant purified InlK were used as a positive control . D . Detection of secreted InlK in the supernatant of ΔsrtA mutants over - expressing InlK . Western blotting was carried out on trichloroacetic acid precipitates of ΔsrtA and ΔsrtA + pPRT - inlK culture ( OD600 = 1 ) supernatants using the rabbit polyclonal anti - InlK antibody . E . Detection of purified recombinant InlK protein with rabbit polyclonal anti - live Listeria antibody , rabbit polyclonal anti - killed Listeria antibody , rabbit polyclonal anti - InlK and a rabbit pre - immune serum . InlK was detected only with the rabbit polyclonal anti - live Listeria antibody indicating that it is expressed during the in vivo infectious process . BSA was used as control protein . Two different amounts of proteins were tested ( 500 ng and 200 ng ) to access signal specificity .

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

Figure 2A. InlK amino acid sequence. The signal sequence is underlined and the different regions of leucine rich repeats (LRRs) are outlined. The consensus pentapeptide LPXTG at the C-terminal end is boxed.B. Detection by immunofluorescence microscopy of InlK over-expressing in L. monocytogenes EGD-e (WT), ΔinlK, WT+pADc-inlK, ΔinlK+pPRT-inlK and the ΔsrtA mutant over-expressing inlK (ΔsrtA+pPRT-inlK) grown in BHI medium using the rabbit polyclonal anti-InlK antibody. InlK was detected at the surface of InlK over-expressing bacteria (WT+pADc-inlK and ΔinlK+pPRT-inlK), whereas it was undetectable at the surface WT bacteria or at the surface of the ΔstrA mutant over-expressing inlK.C. Detection of InlK by Western blot on total lysates of L. monocytogenes EGD-e (WT), ΔinlK and ΔinlK+pPRT-inlK grown in BHI using the rabbit polyclonal anti-InlK antibody. Decreased concentrations of recombinant purified InlK were used as a positive control.D. Detection of secreted InlK in the supernatant of ΔsrtA mutants over-expressing InlK. Western blotting was carried out on trichloroacetic acid precipitates of ΔsrtA and ΔsrtA+pPRT-inlK culture (OD600 = 1) supernatants using the rabbit polyclonal anti-InlK antibody.E. Detection of purified recombinant InlK protein with rabbit polyclonal anti-live Listeria antibody, rabbit polyclonal anti-killed Listeria antibody, rabbit polyclonal anti-InlK and a rabbit pre-immune serum. InlK was detected only with the rabbit polyclonal anti-live Listeria antibody indicating that it is expressed during the in vivo infectious process. BSA was used as control protein. Two different amounts of proteins were tested (500 ng and 200 ng) to access signal specificity.


Figure 3A . Bacterial pull - down of purified GST - MVP with the L . monocytogenes strains WT + pPRT - empty , ΔinlK + pPRT - empty , ΔinlK + pPRT - inlK and WT + pPRT - inlJ . GST - MVP bound to InlK over - expressing bacteria ( ΔinlK + pPRT - inlK ) but not to other bacteria . GST - ScarA was used as control of the specificity of the MVP precipitation by InlK over - expressing bacteria . B . Bacterial pull - down of MVP - GFP from transfected HeLa cell lysates with the L . monocytogenes strains WT + pPRT - empty , ΔinlK + pPRT - empty , ΔinlK + pPRT - inlK and WT + pPRT - inlJ . MVP - GFP bound to InlK over - expressing bacteria ( ΔinlK + pPRT - inlK ) but not to other bacteria . C . Co - immunoprecipitation ( Co - IP ) of purified InlK ( 20 µg ) with endogenous MVP of HT29 cells . The right panel shows anti - MVP co - IP performed on HT29 cell lysates . The left panel shows the control anti - MVP co - IP performed on lysis buffer . D . Detection of MVP recruitment at the surface of InlK over - expressing bacteria . HeLa cells were transfected with MVP - GFP ( green ) , infected with L . monocytogenes EGD - e wild - type ( WT ) , ΔinlK or ΔinlK + pPRT - inlK for 4 h , fixed for fluorescence light microscopy , and stained with anti - Listeria antibodies ( red ) . Inset regions are magnified . The scale bar represents 1 µm .

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

Figure 3A. Bacterial pull-down of purified GST-MVP with the L. monocytogenes strains WT+pPRT-empty, ΔinlK+pPRT-empty, ΔinlK+pPRT-inlK and WT+pPRT-inlJ. GST-MVP bound to InlK over-expressing bacteria (ΔinlK+pPRT-inlK) but not to other bacteria. GST-ScarA was used as control of the specificity of the MVP precipitation by InlK over-expressing bacteria.B. Bacterial pull-down of MVP-GFP from transfected HeLa cell lysates with the L. monocytogenes strains WT+pPRT-empty, ΔinlK+pPRT-empty, ΔinlK+pPRT-inlK and WT+pPRT-inlJ. MVP-GFP bound to InlK over-expressing bacteria (ΔinlK+pPRT-inlK) but not to other bacteria.C. Co-immunoprecipitation (Co-IP) of purified InlK (20 µg) with endogenous MVP of HT29 cells. The right panel shows anti-MVP co-IP performed on HT29 cell lysates. The left panel shows the control anti-MVP co-IP performed on lysis buffer.D. Detection of MVP recruitment at the surface of InlK over-expressing bacteria. HeLa cells were transfected with MVP-GFP (green), infected with L. monocytogenes EGD-e wild-type (WT), ΔinlK or ΔinlK+pPRT-inlK for 4 h, fixed for fluorescence light microscopy, and stained with anti-Listeria antibodies (red). Inset regions are magnified. The scale bar represents 1 µm.


Figure 4A . Detection of MVP recruitment at the surface of intracellular InlK over - expressing bacteria . HeLa cells were transfected with MVP - GFP ( red ) , infected with InlK expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for fluorescence light microscopy . Intra - ( only green ) and extracellular ( cyan = green + blue ) bacteria were differentially stained with anti - Listeria antibody ( cf Material and Methods ) . Inset regions are magnified . Arrows indicate another intracellular bacterium which recruit MVP - GFP . The scale bar represents 1 µm . The right panel represents the quantification of the intracellular bacteria that recruit MVP ( mean % ± SEM % ) shown in the left panel . Statistical analyses were performed on the results of 3 independent experiments using the Student ' s t test . No significant difference was found between the 4 time points . B . Detection of MVP recruitment at the surface of intracytosolic InlK over - expressing bacteria . HeLa cells were transfected with MVP - tomato ( red ) and YFP - CBD ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for fluorescence light microscopy and stained with phalloidin ( blue ) . MVP positive bacteria were also labeled with YFP - CBD revealing that MVP was recruited by intracytosolic bacteria after the lysis of the internalization vacuole . Inset regions are magnified . The scale bar represents 1 µm . C . Kinetics of MVP and actin recruitment at the surface of InlK over - expressing bacteria . HeLa cells were transfected with MVP - tomato ( red ) and actin - GFP ( 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 15 min for 2 h . The left part shows an MVP positive bacterium that never recruits actin . The right part shows MVP replacement by actin around the bacterium . No colocalization of MVP - Tomato and actin - GFP was detected . Time is indicated along the Y axis . The entire image sequence can be viewed as Video S1 .

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

Figure 4A. Detection of MVP recruitment at the surface of intracellular InlK over-expressing bacteria. HeLa cells were transfected with MVP-GFP (red), infected with InlK expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy. Intra- (only green) and extracellular (cyan = green+blue) bacteria were differentially stained with anti- Listeria antibody (cf Material and Methods). Inset regions are magnified. Arrows indicate another intracellular bacterium which recruit MVP-GFP. The scale bar represents 1 µm. The right panel represents the quantification of the intracellular bacteria that recruit MVP (mean%±SEM%) shown in the left panel. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. No significant difference was found between the 4 time points.B. Detection of MVP recruitment at the surface of intracytosolic InlK over-expressing bacteria. HeLa cells were transfected with MVP-tomato (red) and YFP-CBD (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy and stained with phalloidin (blue). MVP positive bacteria were also labeled with YFP-CBD revealing that MVP was recruited by intracytosolic bacteria after the lysis of the internalization vacuole. Inset regions are magnified. The scale bar represents 1 µm.C. Kinetics of MVP and actin recruitment at the surface of InlK over-expressing bacteria. HeLa cells were transfected with MVP-tomato (red) and actin-GFP (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 15 min for 2 h. The left part shows an MVP positive bacterium that never recruits actin. The right part shows MVP replacement by actin around the bacterium. No colocalization of MVP-Tomato and actin-GFP was detected. Time is indicated along the Y axis. The entire image sequence can be viewed as Video S1.


Figure 5A . Impaired recruitment of p62 to MVP positive Listeria . HeLa cells were transfected with MVP - GFP ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for fluorescence light microscopy , and stained with phalloidin ( blue ) and anti - p62 antibody ( red ) . Inset regions are magnified . Arrows indicate independent bacteria The scale bar represents 1 µm . The vast majority of MVP - positive bacteria were completely devoid of anti - p62 labeling ( 95 . 1 ± 2 . 0 % ; mean ± SEM from n = 3 experiments ) but 4 . 9 ± 2 . 0 % ( mean ± SEM from n = 3 experiments ) were stained at one pole with MVP and at the other pole with p62 . 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 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 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 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 .

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

Figure 5A. Impaired recruitment of p62 to MVP positive Listeria. HeLa cells were transfected with MVP-GFP (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue) and anti-p62 antibody (red). Inset regions are magnified. Arrows indicate independent bacteria The scale bar represents 1 µm. The vast majority of MVP-positive bacteria were completely devoid of anti-p62 labeling (95.1±2.0%; mean ± SEM from n = 3 experiments) but 4.9±2.0% (mean ± SEM from n = 3 experiments) were stained at one pole with MVP and at the other pole with p62.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 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 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 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.


Figure 6A . 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 ) 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 ) 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 ) 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 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 ) 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 * .

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

Figure 6A. 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) 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) 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) 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 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) 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 *.


Figure 1A 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 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 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 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 .

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

Figure 1A 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 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 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 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.


Figure 2A 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 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 / + 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 ) .

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

Figure 2A 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 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/+ 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).


Figure 3A , 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 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 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 .

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

Figure 3A, 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, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.C, D The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing Pdp1ε (C) or carrying heterozygous Pdp1ε3135 mutation (D). Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$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 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.


Figure 4A 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 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 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 .

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

Figure 4A 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 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 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.


Figure 5A 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 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 ) .

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

Figure 5A 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 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).


Figure 6A , 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 ) and control ( timG4 / + ; Udcr2 / + ) collected on DD1 ( n = 5 ) . 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 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 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 .

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

Figure 6A, 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, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.C Plots of relative mRNA abundance vs CT for clock genes determined by qRT-PCR in whole head extracts of not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) collected on DD1 (n=5).D ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim and Pdp1ε in not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) (n=3). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical difference is measured using two-way ANOVA, P<0.001, significant effect of genotypes were found for tim E-box (P<0.01), Pdp1ε E-box (P<0.05), tim TSS (P<0.05) and Pdp1ε TSS (P<0.001) and tim gene body (P<0.01). G4, GAL4; U, UAS.E, F The period of DD locomotor rhythm of flies when knocking down Nipped-A and over-expressing not. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$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 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.


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

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

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


Figure 2 ( 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 ) 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 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 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 .

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

Figure 2(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) 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 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 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.


Figure 3 ( 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 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 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 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 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 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 .

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

Figure 3(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 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 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 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 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 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.


Figure 4 ( 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 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 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 .

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

Figure 4(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 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 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.


Figure 1B 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 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 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 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 .

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Figure 1B 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 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 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 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.


Figure 2A 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 . 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 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 ) .

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Figure 2A 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.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 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).


Figure 3C 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 .

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Figure 3C 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.


Figure 4D 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 .

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Figure 4D 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.


Figure 5A 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 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 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 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 .

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Figure 5A 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 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 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 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.


Figure 6A 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 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 .

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Figure 6A 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 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.


Figure 1 . 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 .

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Figure 1.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.


Figure 2 . 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 .

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Figure 2.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.


Figure 3 . 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 .

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Figure 3.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.


Figure 4 . 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 .

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

Figure 4.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.


Figure 5 . 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 .

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

Figure 5.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.