F . Fraction of Ptc positive that overlaps with Atg8 in germaria of the genotypes shown in E . n number is indicated in E . An unpaired parametric Welch ' s t - test was used to establish the statistical significance ( * * * p ≤ 0 . 001 ) . The Y axis in the graph shows the Manders ' Coefficient M1 - M2 . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

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

F. Fraction of Ptc positive that overlaps with Atg8 in germaria of the genotypes shown in E. n number is indicated in E. An unpaired parametric Welch's t-test was used to establish the statistical significance (*** p≤0.001). The Y axis in the graph shows the Manders' Coefficient M1-M2. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


G . Fraction of Atg8 + autophagosomes that overlaps with Ptc in germaria of the genotypes shown in E . n number is indicated in E . An unpaired parametric Welch ' s t - test was used to establish the statistical significance ( * * p ≤ 0 . 01 ) . The Y axis in the graph shows the Manders ' Coefficient M2 - M1 . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

G. Fraction of Atg8+ autophagosomes that overlaps with Ptc in germaria of the genotypes shown in E. n number is indicated in E. An unpaired parametric Welch's t-test was used to establish the statistical significance (** p≤0.01). The Y axis in the graph shows the Manders' Coefficient M2-M1. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


B . Total number of Atg8 + autophagosomes in germaria of the following genotypes ptc - Gal4 ( BL2017 ) / UAS - GFP - mCherry - Atg8 ( BL37749 ) ( n = 21 ) . ptc - Gal4 ( BL2017 ) / UAS - GFP - mCherry - Atg8 ( BL37749 ) ; DroncKOTub - G80ts ( BL7019 ) / + ( n = 28 ) . An unpaired parametric Welch ' s t - test was used to establish the statistical significance ( * p ≤ 0 . 05 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Total number of Atg8+ autophagosomes in germaria of the following genotypes ptc-Gal4 (BL2017)/ UAS-GFP-mCherry-Atg8 (BL37749) (n=21). ptc-Gal4 (BL2017)/ UAS-GFP-mCherry-Atg8 (BL37749); DroncKOTub-G80ts (BL7019)/+ (n=28). An unpaired parametric Welch's t-test was used to establish the statistical significance (* p≤0.05). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


C . Volume of Atg8 + autophagosomes in germaria of the genotypes described in B . n number is indicated in B . A non - parametric Mann - Whitney t - test was used to determine statistical significance ( * * * p < 0 . 001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

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

C. Volume of Atg8+ autophagosomes in germaria of the genotypes described in B. n number is indicated in B. A non-parametric Mann-Whitney t-test was used to determine statistical significance (*** p<0.001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


H . Western blot showing the expression levels of the autophagy marker p62 ( upper lane ) , Caspase - 9 ( middle lane ) and Actin ( bottom lane , loading control ) in either scrambled or Caspase - 9 deficient OVCAR - 3 cells ; the protein levels of the different read outs were measured at 72h after siRNA treatment in cells grown during the last 4 h before sample processing in our standard cell culture conditions , in cell culture media containing EtOH ( 0 . 2 % ) , and in cell culture media containing EtOH ( 0 . 2 % ) + bafilomycin A1 ( 400nM ) .

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

H. Western blot showing the expression levels of the autophagy marker p62 (upper lane), Caspase-9 (middle lane) and Actin (bottom lane, loading control) in either scrambled or Caspase-9 deficient OVCAR-3 cells; the protein levels of the different read outs were measured at 72h after siRNA treatment in cells grown during the last 4 h before sample processing in our standard cell culture conditions, in cell culture media containing EtOH (0.2%), and in cell culture media containing EtOH (0.2%) + bafilomycin A1 (400nM).


I . Quantification of p62 protein levels in the experimental conditions described in H . one sample T Wilcoxon test was used to calculate statistical significance , * p ≤ 0 . 05 , N ≥ 3 . Bars indicate value of the mean while error bars represent the standard deviation .

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

I. Quantification of p62 protein levels in the experimental conditions described in H. one sample T Wilcoxon test was used to calculate statistical significance, * p≤0.05, N≥3. Bars indicate value of the mean while error bars represent the standard deviation.


K . Quantification of the total number of Castor - expressing cells within the FasIII cellular domain in the genotypes described in J . n number is indicated also in J . An ordinary one - way ANOVA and Dunnett ' s multiple comparison post - test were used to determine statistical significance ( n . s . = not significant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

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

K. Quantification of the total number of Castor-expressing cells within the FasIII cellular domain in the genotypes described in J. n number is indicated also in J. An ordinary one-way ANOVA and Dunnett's multiple comparison post-test were used to determine statistical significance (n.s.= not significant, ** p≤0.01, **** p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


( C , D Confocal micrographs of HeLa cells expressing GFP - TECPR1 and treated with osmotic shock assay or LLOMe ( C ) , or infected with the bacteria indicated ( D ) and fixed at 1 h ( S . Typhimurium and L . monocytogenes ) or 0 . 5 h ( S . flexneri ) post - infection . Cells were stained for Galectin - 8 and DNA ( DAPI ) . Scale bar , 20 μm .

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

(C, D Confocal micrographs of HeLa cells expressing GFP-TECPR1 and treated with osmotic shock assay or LLOMe (C), or infected with the bacteria indicated (D) and fixed at 1 h (S. Typhimurium and L. monocytogenes) or 0.5 h (S. flexneri) post-infection. Cells were stained for Galectin-8 and DNA (DAPI). Scale bar, 20 μm.


( F ) Percentage of bacteria positive for mCherry - TECPR1 in ATG5 knockout ( KO ) or ATG5 - complemented MEF cells at 1 h ( S . Typhimurium ) or 0 . 5 h ( S . flexneri ) post - infection . Mean + / - SD of four ( S . Typhimurium ) or two ( S . flexneri ) independent experiments . n > 100 bacteria per coverslip . ( G ) Percentage of S . Typhimurium positive for GFP - TECPR1 at 1 h post - infection in HeLa cells pretreated or not with 100 nM Wortmannin . Mean + / - SD of two independent experiments . n > 100 bacteria per coverslip .

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

(F) Percentage of bacteria positive for mCherry-TECPR1 in ATG5 knockout (KO) or ATG5-complemented MEF cells at 1 h (S. Typhimurium) or 0.5 h (S. flexneri) post-infection. Mean +/- SD of four (S. Typhimurium) or two (S. flexneri) independent experiments. n > 100 bacteria per coverslip. (G) Percentage of S. Typhimurium positive for GFP-TECPR1 at 1 h post-infection in HeLa cells pretreated or not with 100 nM Wortmannin. Mean +/- SD of two independent experiments. n > 100 bacteria per coverslip.


H ) Confocal micrographs of HeLa cells expressing GFP - TECPR1 either alone or together with FLAG - tagged neutral sphingomyelinase 2 ( nSMase2 ; ( H ) ) Cells were stained for Galectin - 8 and DNA ( DAPI ) . Scale bar , 20 μm . ( I ) Percentage of S . Typhimurium positive for GFP - TECPR1 or endogenous Galectin - 8 at 1 h post - infection in HeLa cells expressing FLAG - nSMase2 or not . Mean + / - SD of two ( TECPR1 ) or three ( Gal8 ) independent experiments . n > 100 bacteria per coverslip .

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

H) Confocal micrographs of HeLa cells expressing GFP-TECPR1 either alone or together with FLAG-tagged neutral sphingomyelinase 2 (nSMase2; (H)) Cells were stained for Galectin-8 and DNA (DAPI). Scale bar, 20 μm. (I) Percentage of S. Typhimurium positive for GFP-TECPR1 or endogenous Galectin-8 at 1 h post-infection in HeLa cells expressing FLAG-nSMase2 or not. Mean +/- SD of two (TECPR1) or three (Gal8) independent experiments. n > 100 bacteria per coverslip.


( C ) Percentage of S . Typhimurium positive for the indicated constructs in HeLa cells , fixed at the indicated timepoints post - infection . Mean + / - SEM of 3 - 4 independent experiments performed in duplicate . n > 100 bacteria per coverslip .

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

(C) Percentage of S. Typhimurium positive for the indicated constructs in HeLa cells, fixed at the indicated timepoints post-infection. Mean +/- SEM of 3-4 independent experiments performed in duplicate. n > 100 bacteria per coverslip.


( E ) Confocal micrographs of HeLa cells expressing TECPR1 N ' DysF - GFP with or without FLAG - nSMase2 , infected with S . Typhimurium , fixed at 30 min post - infection and stained with anti - Galectin 8 antibody . DNA stained with DAPI . Scale bar , 20 μm .

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

(E) Confocal micrographs of HeLa cells expressing TECPR1 N' DysF-GFP with or without FLAG-nSMase2, infected with S. Typhimurium, fixed at 30 min post-infection and stained with anti-Galectin 8 antibody. DNA stained with DAPI. Scale bar, 20 μm.


( F ) Percentage of S . Typhimurium positive for N ' DysF - GFP in Hela cells , expressing FLAG - nSMase or not , fixed at 30 min post - infection . Mean + / - SEM of three independent experiments performed in duplicate .

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

(F) Percentage of S. Typhimurium positive for N' DysF-GFP in Hela cells, expressing FLAG-nSMase or not, fixed at 30 min post-infection. Mean +/- SEM of three independent experiments performed in duplicate.


( G and H ) Confocal micrographs of HeLa cells expressing the indicated GFP - tagged DysF domains , infected with mCherry - S . Typhimurium and fixed at 30 min post - infection ( G ) or treated with osmotic shock assay , fixed and stained with anti - Galectin 8 antibody ( H ) . The bottom row in H depicts an enlarged version of the boxed region shown above . Scale bar , 20 μm .

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

(G and H) Confocal micrographs of HeLa cells expressing the indicated GFP-tagged DysF domains, infected with mCherry-S. Typhimurium and fixed at 30 min post-infection (G) or treated with osmotic shock assay, fixed and stained with anti-Galectin 8 antibody (H). The bottom row in H depicts an enlarged version of the boxed region shown above. Scale bar, 20 μm.


( B Percentage of S . Typhimurium positive for the indicated GFP - tagged N - terminal DysF constructs at 30 min post - infection . Mean + / - SD of two ( EK134 , 135AA ) or three independent experiments . n > 100 bacteria per coverslip . p value from one - way ANOVA with Dunnett ' s multiple comparisons test .

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

(B Percentage of S. Typhimurium positive for the indicated GFP-tagged N-terminal DysF constructs at 30 min post-infection. Mean +/- SD of two (EK134,135AA) or three independent experiments. n > 100 bacteria per coverslip. p value from one-way ANOVA with Dunnett's multiple comparisons test.


C ) Percentage of S . Typhimurium positive for the indicated GFP - tagged N - terminal DysF constructs at 30 min post - infection . Mean + / - SD of three independent experiments . n > 100 bacteria per coverslip . p value from one - way ANOVA with Dunnett ' s multiple comparisons test .

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

C) Percentage of S. Typhimurium positive for the indicated GFP-tagged N-terminal DysF constructs at 30 min post-infection. Mean +/- SD of three independent experiments. n > 100 bacteria per coverslip. p value from one-way ANOVA with Dunnett's multiple comparisons test.


( F ) Confocal micrographs of HeLa cells expressing either GFP - TECPR1 WT or W154A and treated with LLOMe or DMSO vehicle control for 10 min and fixed . Scale bar , 20 μm . ( G ) Number of GFP - positive puncta per cell were enumerated in samples depicted in ( F ) . Data from 50 cells from a single experiment , representative of two . p value from one - way ANOVA with Tukey ' s multiple comparisons test .

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

(F) Confocal micrographs of HeLa cells expressing either GFP-TECPR1 WT or W154A and treated with LLOMe or DMSO vehicle control for 10 min and fixed. Scale bar, 20 μm. (G) Number of GFP-positive puncta per cell were enumerated in samples depicted in (F). Data from 50 cells from a single experiment, representative of two. p value from one-way ANOVA with Tukey's multiple comparisons test.


( C - E ) Liposome sedimentation assay . The indicated proteins were incubated with either liposomes ( containing sphingomyelin or not ) or buffer only , pelleted by centrifugation and equal portions of supernatant and pellet fractions analysed by SDS - PAGE and Coomassie staining . Representative gels depicted ( C ) . Fraction of TECPR1 ( D ) or DysF WT or W154A ( E ) proteins present in pellet under the indicated conditions , expressed as % binding . Note pelleting in the absence of liposomes likely due to affinity of proteins for tube walls . Mean + / - SD of three independent experiments . p value from one - way ANOVA with Tukey ' s multiple comparisons test .

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

(C-E) Liposome sedimentation assay. The indicated proteins were incubated with either liposomes (containing sphingomyelin or not) or buffer only, pelleted by centrifugation and equal portions of supernatant and pellet fractions analysed by SDS-PAGE and Coomassie staining. Representative gels depicted (C). Fraction of TECPR1 (D) or DysF WT or W154A (E) proteins present in pellet under the indicated conditions, expressed as % binding. Note pelleting in the absence of liposomes likely due to affinity of proteins for tube walls. Mean +/- SD of three independent experiments. p value from one-way ANOVA with Tukey's multiple comparisons test.


( F ) In vitro LC3 lipid conjugation assay . Liposomes containing sphingomyelin or not were incubated with LC3 lipid conjugation machinery : ATG7 ( E1 ) , ATG3 ( E2 ) , ATG5 - ATG12 ( E3 ) , LC3B , MgCl2 , ATP and the indicated TECPR1 proteins . Control reactions without ATG3 or ATP were included . Mixes were incubated at 37 C for 3 h , centrifuged and equal fractions of both supernatant and pellet analysed by SDS - PAGE and Coomassie staining . Lipidated LC3B - II appears in pellet fraction whereas both soluble LC3B - I and AMPylated LC3B are in the supernatant , the latter of the same apparent molecular weight as LC3B - II .

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

(F) In vitro LC3 lipid conjugation assay. Liposomes containing sphingomyelin or not were incubated with LC3 lipid conjugation machinery: ATG7 (E1), ATG3 (E2), ATG5-ATG12 (E3), LC3B, MgCl2, ATP and the indicated TECPR1 proteins. Control reactions without ATG3 or ATP were included. Mixes were incubated at 37 C for 3 h, centrifuged and equal fractions of both supernatant and pellet analysed by SDS-PAGE and Coomassie staining. Lipidated LC3B-II appears in pellet fraction whereas both soluble LC3B-I and AMPylated LC3B are in the supernatant, the latter of the same apparent molecular weight as LC3B-II.


( G ) Fraction of LC3B protein present in pellet from complete in vitro LC3 lipid conjugation assay with the indicated TECPR1 proteins in either absence or presence of sphingomyelin . Mean + / - SD from three independent experiments . p value from one - way ANOVA with Tukey ' s multiple comparisons test .

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

(G) Fraction of LC3B protein present in pellet from complete in vitro LC3 lipid conjugation assay with the indicated TECPR1 proteins in either absence or presence of sphingomyelin. Mean +/- SD from three independent experiments. p value from one-way ANOVA with Tukey's multiple comparisons test.


( A Confocal micrographs of indicated MEF cell lines infected with mCherry - expressing S . Typhimurium , fixed at 30 min post - infection and stained with anti - ATG5 antibody ( A ) Scale bar , 20 μm .

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

(A Confocal micrographs of indicated MEF cell lines infected with mCherry-expressing S. Typhimurium, fixed at 30 min post-infection and stained with anti-ATG5 antibody (A) Scale bar, 20 μm.


C ) Confocal micrographs of indicated MEF cell lines infected with mCherry - expressing S . Typhimurium , fixed at 30 min post - infection and stained with anti - LC3 antibody ( C ) . Scale bar , 20 μm .

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

C) Confocal micrographs of indicated MEF cell lines infected with mCherry-expressing S. Typhimurium, fixed at 30 min post-infection and stained with anti-LC3 antibody (C). Scale bar, 20 μm.


( E ) Percentage of S . Typhimurium positive for endogenous LC3 in indicated MEF cells treated with 100 nM Wortmannin or DMSO vehicle control , fixed at 1 h post - infection . Mean + / - SEM of three independent experiments performed in duplicate . n > 100 bacteria per coverslip . p value from one - way ANOVA with Tukey ' s multiple comparisons test .

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

(E) Percentage of S. Typhimurium positive for endogenous LC3 in indicated MEF cells treated with 100 nM Wortmannin or DMSO vehicle control, fixed at 1 h post-infection. Mean +/- SEM of three independent experiments performed in duplicate. n > 100 bacteria per coverslip. p value from one-way ANOVA with Tukey's multiple comparisons test.


( F ) Confocal micrographs of either control of ATG16L1 - deficient MEF cells , transfected with either control or TECPR1 siRNA ( siRNA no . 66 depicted ) , infected with mCherry - expressing S . Typhimurium for 30 min , fixed and stained with anti - LC3 antibody . Scale bar , 20 μm .

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

(F) Confocal micrographs of either control of ATG16L1-deficient MEF cells, transfected with either control or TECPR1 siRNA (siRNA no. 66 depicted), infected with mCherry-expressing S. Typhimurium for 30 min, fixed and stained with anti-LC3 antibody. Scale bar, 20 μm.


( A ) Percentage of S . Typhimurium positive for Galectin - 8 and / or GFP - TECPR1 in either control Hela cells or those expressing GFP - TECPR1 at 1 h post - infection . Mean + / - SEM of three independent experiments performed in duplicate . n > 100 bacteria per coverslip . ( B ) Confocal micrographs of HeLa cells expressing the indicated GFP - TECPR1 constructs or not ( control ) , infected with S . Typhimurium for 30 min , fixed and stained with anti - Galectin - 8 and DAPI . Scale bar , 20 μm . Arrowheads indicate bacteria positive for Galectin 8 , of which more are present in cells expressing GFP - TECPR1 WT but not W154A . Arrow indicates bacterium to which GFP - TECPR1 but not Galectin 8 has been recruited .

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

(A) Percentage of S. Typhimurium positive for Galectin-8 and/or GFP-TECPR1 in either control Hela cells or those expressing GFP-TECPR1 at 1 h post-infection. Mean +/- SEM of three independent experiments performed in duplicate. n > 100 bacteria per coverslip. (B) Confocal micrographs of HeLa cells expressing the indicated GFP-TECPR1 constructs or not (control), infected with S. Typhimurium for 30 min, fixed and stained with anti-Galectin-8 and DAPI. Scale bar, 20 μm. Arrowheads indicate bacteria positive for Galectin 8, of which more are present in cells expressing GFP-TECPR1 WT but not W154A. Arrow indicates bacterium to which GFP-TECPR1 but not Galectin 8 has been recruited.


( C ) Percentage of S . Typhimurium positive for Galectin - 8 in Hela cells expressing the indicated GFP - TECPR1 constructs or not ( control ) and infected with S . Typhimurium for 1 h . Mean + / - SEM of five independent experiments performed in triplicate . p value from one - way ANOVA with Tukey ' s multiple comparisons test .

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

(C) Percentage of S. Typhimurium positive for Galectin-8 in Hela cells expressing the indicated GFP-TECPR1 constructs or not (control) and infected with S. Typhimurium for 1 h. Mean +/- SEM of five independent experiments performed in triplicate. p value from one-way ANOVA with Tukey's multiple comparisons test.


( D ) Fold intracellular proliferation of S . Typhimurium in HeLa cells expressing the indicated GFP - TECPR1 constructs or not ( control ) as assessed by colony forming unit assay . Mean + / - SEM of four ( ΔPH ) or five ( control , WT , W154A and ΔAIR ) independent experiments . p value from one - way ANOVA with Dunnett ' s multiple comparisons test .

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(D) Fold intracellular proliferation of S. Typhimurium in HeLa cells expressing the indicated GFP-TECPR1 constructs or not (control) as assessed by colony forming unit assay. Mean +/- SEM of four (ΔPH) or five (control, WT, W154A and ΔAIR) independent experiments. p value from one-way ANOVA with Dunnett's multiple comparisons test.


( A - B ) Capsids for WT , K158A , K158A / T8I and T8I were classified into the indicated categories . Representative tomograms of K158A mutant ( A ) and the percentage ( B ) for each category are shown . The margin of error was calculated Images and quantification are from the same samples .

(A-B) Capsids for WT, K158A, K158A/T8I and T8I were classified into the indicated categories. Representative tomograms of K158A mutant (A) and the percentage (B) for each category are shown. The margin of error was calculated Images and quantification are from the same samples.


( C ) Virion stability assay using TIRF microscopy to quantify the number of viral particles in presence of SLO and IP6 for the indicated mutant after 1 hour . Mean data represent n = 2 technical replicates . Scale bar 10 μm .

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(C) Virion stability assay using TIRF microscopy to quantify the number of viral particles in presence of SLO and IP6 for the indicated mutant after 1 hour. Mean data represent n=2 technical replicates. Scale bar 10 μm.


( F ) Quantification of CXCL10 and IFIT1 transcripts by qPCR of the indicated THP - 1 cell lines stimulated for 24 hours with LPS ( 1 ng / ml ) normalised to mock . Mean data n = 2 biological replicates .

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(F) Quantification of CXCL10 and IFIT1 transcripts by qPCR of the indicated THP-1 cell lines stimulated for 24 hours with LPS (1 ng/ml) normalised to mock. Mean data n=2 biological replicates.


( H ) Quantification of the indicated chemokine transcripts by qPCR from PMA - treated THP - 1 / SAMHD1 KO cells transduced for 24 hours with 50ng / RT of the indicated HIV - 1 GFP capsids mutants normalised to mock infected . Mean data represent n = 3 biological replicates .

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(H) Quantification of the indicated chemokine transcripts by qPCR from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants normalised to mock infected. Mean data represent n=3 biological replicates.


( I - J ) Protein quantification of CXCL10 ( I ) and TNFa ( J ) by Luminex ™ from supernatant of PMA - treated THP - 1 / SAMHD1 KO cells infected with the indicated HIV - 1 mutants . Data were normalised to mock . Mean data represent n = 2 biological replicates .

(I-J) Protein quantification of CXCL10 (I) and TNFa (J) by Luminex™ from supernatant of PMA-treated THP-1/SAMHD1 KO cells infected with the indicated HIV-1 mutants. Data were normalised to mock. Mean data represent n=2 biological replicates.


( D ) qPCR quantification of the indicated chemokine transcripts from PMA - treated THP - 1 / SAMHD1 KO cells transduced for 24 hours with the indicated VLPs mutants . ( E ) qPCR quantification of the indicated chemokine transcripts level from PMA - treated THP - 1 / SAMHD1 KO cells transduced for 24 hours with 50ng / RT of the indicated HIV - 1 GFP capsids mutants .

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(D) qPCR quantification of the indicated chemokine transcripts from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with the indicated VLPs mutants. (E) qPCR quantification of the indicated chemokine transcripts level from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants.


( F ) Protein quantification of IL - 6 by Luminex ™ from PMA - treated THP - 1 / SAMHD1 KO cells infected with the indicated HIV - 1 mutants .

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(F) Protein quantification of IL-6 by Luminex™ from PMA-treated THP-1/SAMHD1 KO cells infected with the indicated HIV-1 mutants.


IFIT1 - Luc reporter activity from PMA - treated THP - 1 / SAMHD1 KO cells stimulated by transfection with either poly I : C ( 0 . 5 μg / ml ) ( B ) mean data represent n = 2 biological

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IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection with either poly I:C (0.5 μg/ml) (B) mean data represent n=2 biological replicates


IFIT1 - Luc reporter activity from PMA - treated THP - 1 / SAMHD1 KO cells stimulated by transfection with HT - DNA ( 0 . 1 μg / ml ) ( C ) mean data represent n = 2 biological

IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection with HT-DNA (0.1 μg/ml) (C) mean data represent n=2 biological replicates


IFIT1 - Luc reporter activity from PMA - treated THP - 1 / SAMHD1 KO cells stimulated by transfection infected for 24 hours with 50ng / RT of the indicated HIV - 1 GFP capsids mutants ( D ) .

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IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection infected for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants (D).


IFIT1 - Luc reporter activity from PMA - treated THP - 1 / SAMHD1 KO infected with for 24 hours with 50ng / RT of the indicated HIV - 1 GFP capsids mutants in the presence of DMSO or 0 . 5 μg / ml H151 ( E )

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IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO infected with for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants in the presence of DMSO or 0.5 μg/ml H151 (E)


( B - D ) Protein quantification of CXCL10 ( B ) , IL - 6 ( C ) and TNFa ( D ) by Luminex ™ from HMDMs infected with the indicated HIV - 1 mutants .

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(B-D) Protein quantification of CXCL10 (B), IL-6 (C) and TNFa (D) by Luminex™ from HMDMs infected with the indicated HIV-1 mutants.


( F - H ) Protein quantification from SupT1 cells as in ( B - D ) ) .

(F-H) Protein quantification from SupT1 cells as in (B-D)).


Protein quantification from infected Jurkat T cells as in ( B - D ) .

Protein quantification from infected Jurkat T cells as in (B-D).


B ) Volcano plot for the F test on differential expression between WT and mdx mice . C ) Volcano plot for the F test on differential expression between WT and mdx + + mice .

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B) Volcano plot for the F test on differential expression between WT and mdx mice. C) Volcano plot for the F test on differential expression between WT and mdx++ mice.


D ) Volcano plot for the F test on differential expression between WT and mdx + - mice .

D) Volcano plot for the F test on differential expression between WT and mdx+- mice.


E , F ) Boxplot comparing the distribution of the Dmd and Prune2 genes in the WT , mdx , mdx + + and mdx + - mice groups . The central band of the boxplot denotes the median ; the box denotes the first and third quartile ; the whiskers are computed as min ( max ( x ) , Q3 + 1 . 5 IQR ) and max ( min ( x ) , Q1 - 1 . 5 * IQR ) , where min ( x ) and max ( x ) denote the minimum and maximum of the distribution , Q1 and Q3 the first and third quartile , and IQR the interquartile range .

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E, F) Boxplot comparing the distribution of the Dmd and Prune2 genes in the WT, mdx, mdx++ and mdx+- mice groups. The central band of the boxplot denotes the median; the box denotes the first and third quartile; the whiskers are computed as min(max(x), Q3 + 1.5 IQR) and max(min(x), Q1 - 1.5 * IQR), where min(x) and max(x) denote the minimum and maximum of the distribution, Q1 and Q3 the first and third quartile, and IQR the interquartile range.


G ) Volcano plot for the F tests on differential expression between mdx + + and mdx + - mice .

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G) Volcano plot for the F tests on differential expression between mdx++ and mdx+- mice.


Scatter plots comparing the estimated logFCs across mice groups . Panel H : mdx vs mdx + + .

Scatter plots comparing the estimated logFCs across mice groups. Panel H: mdx vs mdx++.


Scatter plots comparing the estimated logFCs across mice groups . + + . Panel I : mdx vs mdx + - . Panel J : mdx + + vs mdx + - .

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Scatter plots comparing the estimated logFCs across mice groups. ++. Panel I: mdx vs mdx+-. Panel J: mdx++ vs mdx+-.


A ) Histogram of the likelihood ratio p - values for the test on differences between WT and mdx at any time point .

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A) Histogram of the likelihood ratio p-values for the test on differences between WT and mdx at any time point.


B ) Distribution of the estimated logFCs for mdx versus WT mice at weeks 6 , 12 , 18 , 24 and 30 for the 1532 genes with significant differences between WT and mdx .

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B) Distribution of the estimated logFCs for mdx versus WT mice at weeks 6, 12, 18, 24 and 30 for the 1532 genes with significant differences between WT and mdx.


D ) Trajectory plots comparing the trajectories of Chordc1 and Psat1 in WT and mdx mice .

D) Trajectory plots comparing the trajectories of Chordc1 and Psat1 in WT and mdx mice.


E ) Trajectory plots comparing the trajectories of Cd59a , Chordc1 , Hpgd , Ldhb , Lincred1 , Myo6 , Slc39a11 and Tlr1 in WT , mdx + + and mdx + - mice .

E) Trajectory plots comparing the trajectories of Cd59a, Chordc1, Hpgd, Ldhb, Lincred1, Myo6, Slc39a11 and Tlr1 in WT, mdx++ and mdx+- mice.


F ) Scatter plots comparing the estimated logFC of mdx + + vs WT ( x axis ) and of mdx + - vs WT ( y axis ) at weeks 6 , 12 , 18 , 24 and 30 .

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F) Scatter plots comparing the estimated logFC of mdx++ vs WT (x axis) and of mdx+- vs WT (y axis) at weeks 6, 12, 18, 24 and 30.


H ) upstream regulator analysis ( H ) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice .

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H) upstream regulator analysis (H) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice.


A ) Barplot showing the percentage of significant genes in the muscle modules . Next to each module name we report the total number of genes that it comprises . B ) Barplot showing the percentage of significant genes in the blood modules . Next to each module name we report the total number of genes that it comprises . C ) Matrix with the values of the cross - correlation between blood and muscle module eigengenes . To enhance visibility , only correlations with absolute value above 0 . 5 are shown .

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A) Barplot showing the percentage of significant genes in the muscle modules. Next to each module name we report the total number of genes that it comprises. B) Barplot showing the percentage of significant genes in the blood modules. Next to each module name we report the total number of genes that it comprises. C) Matrix with the values of the cross-correlation between blood and muscle module eigengenes. To enhance visibility, only correlations with absolute value above 0.5 are shown.


A ) RT - PCR ( 30 cycles ) showing skipping of exon 23 for PMO and PS49 oligos . No skipping was visible in saline treated mice .

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A) RT-PCR (30 cycles) showing skipping of exon 23 for PMO and PS49 oligos. No skipping was visible in saline treated mice.


B ) Western blot showing the percentage of restoration of dystrophin protein in mdx mice following antisense oligonucleotides administration . M is molecular ladder . WT gastrocnemius represents the standard curve using protein extracts from gastrocnemius muscle . NT is saline treated mdx mice .

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B) Western blot showing the percentage of restoration of dystrophin protein in mdx mice following antisense oligonucleotides administration. M is molecular ladder. WT gastrocnemius represents the standard curve using protein extracts from gastrocnemius muscle. NT is saline treated mdx mice.


C ) Barplot comparing the estimated logFC for the PS49 and PMO treatments in the MoTreat dataset to the desired drug effect obtained from the MoLong dataset ( logFC WT vs mdx at week 12 ) .

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C) Barplot comparing the estimated logFC for the PS49 and PMO treatments in the MoTreat dataset to the desired drug effect obtained from the MoLong dataset (logFC WT vs mdx at week 12).


D , E ) Histograms comparing the distribution of selected genes across different mice groups ( PMO , PS49 and saline in the MoTreat dataset , WT and mdx at week 12 in the MoLong dataset ) .

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D, E) Histograms comparing the distribution of selected genes across different mice groups (PMO, PS49 and saline in the MoTreat dataset, WT and mdx at week 12 in the MoLong dataset).


B ) Volcano plot for the F test on differential expression between healthy individuals and DMD patients .

B) Volcano plot for the F test on differential expression between healthy individuals and DMD patients.


C ) Scatter plot showing the group effect at baseline ( x - axis ) and the effect of the interaction between age and group ( y - axis ) . Black dots represent non differentially expressed genes , while blue dots represent differentially expressed genes .

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C) Scatter plot showing the group effect at baseline (x-axis) and the effect of the interaction between age and group (y-axis). Black dots represent non differentially expressed genes, while blue dots represent differentially expressed genes.


E ) Volcano plot for the F test on differential expression between treated and untreated DMD patients .

E) Volcano plot for the F test on differential expression between treated and untreated DMD patients.


F ) Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the estimates of Liu et al . ( 2012 ) .

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F) Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the estimates of Liu et al. (2012).


G ) Scatter plot showing genes for which a significant effect of treatment with steroids . The group effects at baseline are plotted on the x - axis , while the effects of steroids are plotted on the y - axis .

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G) Scatter plot showing genes for which a significant effect of treatment with steroids. The group effects at baseline are plotted on the x-axis, while the effects of steroids are plotted on the y-axis.


( A - C ) Representative images of Cellular localization of TLR2 - p and scrTLR2 - p peptides in BM - derived macrophages were observed using confocal microscopy . ( A ) Cells were probed with anti TLR2 antibody following by staining with APC - labeled secondary antibody or ( B ) with TLR1 antibody following by staining with FITC - labeled secondary antibody ( left panels , red ) . For membrane staining , cells were labelled with lipid dye , DiD ( C ) . Then rhodamine - labeled fluorescent peptide was added to the cells ( middle panels , green ) . Merged images are shown in the right panel . Scale bars 10µm . The mean Pearson correlation coefficient observed of TLR2 - p or scrTLR2 - p peptides with TLR2 TLR1 and DiD presents as mean of 2 independent experiments ± S . EM ( n = 19 - 61 ) .

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(A-C) Representative images of Cellular localization of TLR2-p and scrTLR2-p peptides in BM-derived macrophages were observed using confocal microscopy. (A) Cells were probed with anti TLR2 antibody following by staining with APC-labeled secondary antibody or (B) with TLR1 antibody following by staining with FITC-labeled secondary antibody (left panels, red). For membrane staining, cells were labelled with lipid dye, DiD (C). Then rhodamine-labeled fluorescent peptide was added to the cells (middle panels, green). Merged images are shown in the right panel. Scale bars 10µm. The mean Pearson correlation coefficient observed of TLR2-p or scrTLR2-p peptides with TLR2 TLR1 and DiD presents as mean of 2 independent experiments ± S.EM (n=19-61).


( A ) Biochemical analyses of peptide interaction with TLR . BM - derived macrophages were incubated with 1µM fluorescently labeled rhodamine ( Rho ) - TLR2 - p or its mutant Rho - ScrTLR2 - p peptide . After 1h of incubation at 370C the cells were lysated . Soluble fraction was used for immunoprecipitation with the indicated TLR antibodies . Proteins samples were resolved by SDS - PAGE and the presence of the labeled peptide was detected with a fluorescent scanner . Subsequently , the gel was transferred to a membrane and subjected to Western blotting for TLR6 , 1 , 2 and 4 in the appropriate samples . Equal loading was measured by detecting of anti - tubulin in the cell lysate . The results are presented as the mean of fluorescent intensity from two independent experiments . Non - specific binding of the peptides to G protein beads was subtracted . The results are mean ± S . E . M of at least two independent experiments .

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(A) Biochemical analyses of peptide interaction with TLR. BM-derived macrophages were incubated with 1µM fluorescently labeled rhodamine (Rho)-TLR2-p or its mutant Rho-ScrTLR2-p peptide. After 1h of incubation at 370C the cells were lysated. Soluble fraction was used for immunoprecipitation with the indicated TLR antibodies. Proteins samples were resolved by SDS-PAGE and the presence of the labeled peptide was detected with a fluorescent scanner. Subsequently, the gel was transferred to a membrane and subjected to Western blotting for TLR6,1,2 and 4 in the appropriate samples. Equal loading was measured by detecting of anti-tubulin in the cell lysate. The results are presented as the mean of fluorescent intensity from two independent experiments. Non-specific binding of the peptides to G protein beads was subtracted. The results are mean± S.E.M of at least two independent experiments.


( B ) Proteins analysis of secreted pro - inflammatory cytokines , IL - 6 and TNF - α from primary CD115 + cells . Cells were incubated with 20µM of TLR2 - p or scrTLR2 - p peptide for 0 . 5h , then washed and incubated with LTA ( 5μg / ml ) , PAM3CSK ( 0 . 5μg / ml ) or LPS ( 0 . 2μg / ml ) at 370C for 5h ( for TNF - α detection ) and 22h ( for IL - 6 detection ) . The results are mean ± S . E . M of two independent experiments ( n = 8 ) .

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(B) Proteins analysis of secreted pro-inflammatory cytokines, IL-6 and TNF-α from primary CD115+ cells. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with LTA (5μg/ml), PAM3CSK (0.5μg/ml) or LPS (0.2μg/ml) at 370C for 5h (for TNF-α detection) and 22h (for IL-6 detection). The results are mean± S.E.M of two independent experiments (n=8).


( B - C ) Represented images of cellular interaction between TLR2 and TLR6 ( A ) and between F4 / 80 and TLR2 ( B ) in BM - derived macrophages with the indicated treatments were observed by Fluorescence Resonance Energy Transfer ( FRET ) using ImageStreamX . Scale bars 10μm . Cells were incubated with 20µM of TLR2 - p or scrTLR2 - p peptide for 0 . 5h , then washed and incubated with 500ng / ml LTA for another 0 . 5h at 370C . Cells were probed with anti TLR6 - PE or anti F4 / 80 - PE conjugated antibody ( donor ) and anti TLR2 antibody following by staining with APC - labeled secondary antibody ( acceptor ) . PE intensity ( middle panel ) and FRET intensity ( right panel ) were measured via ImageStreanX imaging flow cytometer . The FRET intensity shown are mean ± S . E . M of two independent experiments ( n = 2579 - 15567 ) .

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(B-C) Represented images of cellular interaction between TLR2 and TLR6 (A) and between F4/80 and TLR2 (B) in BM-derived macrophages with the indicated treatments were observed by Fluorescence Resonance Energy Transfer (FRET) using ImageStreamX. Scale bars 10μm. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with 500ng/ml LTA for another 0.5h at 370C. Cells were probed with anti TLR6-PE or anti F4/80-PE conjugated antibody (donor) and anti TLR2 antibody following by staining with APC-labeled secondary antibody (acceptor). PE intensity (middle panel) and FRET intensity (right panel) were measured via ImageStreanX imaging flow cytometer. The FRET intensity shown are mean ± S.E.M of two independent experiments (n=2579-15567).


( D ) Represented image of ERK1 , 2 phosphorylation levels in CD115 + BM monocytes cells upon LTA exposure . Cells were pre - treated for 0 . 5h with 20µM of TLR2 - p peptide , scrTLR2 - p peptide or untreated , then washed and incubated with 500ng / ml LTA for the indicated times . ERK1 , 2 phosphorylation levels and total ERK1 , 2 levels were detected by western blotting . Equal loading was detected by measuring anti tubulin . Results are representative data of 2 independent experiments . The bands intensity was quantified for p - ERK levels after 15min of LTA exposure . Results are normalized to tubulin levels and are the mean of 2 experiments ± S . D . M .

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(D) Represented image of ERK1,2 phosphorylation levels in CD115+BM monocytes cells upon LTA exposure. Cells were pre-treated for 0.5h with 20µM of TLR2-p peptide, scrTLR2-p peptide or untreated, then washed and incubated with 500ng/ml LTA for the indicated times. ERK1,2 phosphorylation levels and total ERK1,2 levels were detected by western blotting. Equal loading was detected by measuring anti tubulin. Results are representative data of 2 independent experiments. The bands intensity was quantified for p-ERK levels after 15min of LTA exposure. Results are normalized to tubulin levels and are the mean of 2 experiments ± S.D.M.


( B ) Representative colonoscopy images of the indicated treatments ( day 7 ) . C57BL mice were treated with 2 % of dextran sulfate sodium ( DSS ) in drinking water for 7 days . An amount of 5mg / Kg of TLR2 - p and scrTLR2 - p peptides were injected IP every following day from day one . ( C ) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only , DSS with TLR2 - p peptide , DSS with scrTLR2 - p peptide and untreated mice ( n = 19 , 21 , 22 and 19 respectively ) . Results represent 3 independent experiments , scale bars 100µm and 1000μm .

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(B) Representative colonoscopy images of the indicated treatments (day 7). C57BL mice were treated with 2% of dextran sulfate sodium (DSS) in drinking water for 7 days. An amount of 5mg/Kg of TLR2-p and scrTLR2-p peptides were injected IP every following day from day one.(C) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only, DSS with TLR2-p peptide, DSS with scrTLR2-p peptide and untreated mice (n= 19, 21, 22 and 19 respectively). Results represent 3 independent experiments, scale bars 100µm and 1000μm.


( D ) A graphical summary of changes in body weight ( day 6 ) of the indicated groups .

(D) A graphical summary of changes in body weight (day 6) of the indicated groups.


( E ) Representative H & amp ; amp ; E histological images for distal colon section ( day 7 ) of 9 - 10 sections from 2 independent experiments . Black asterisk indicates on large collection of blood in the lumen , arrowheads indicate on widespread mucosal collapse and ulceration and arrows indicates on edema in the sub mucosa . ( F ) A graphical summary of histological severity score of the indicated treatments .

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(E) Representative H&amp;E histological images for distal colon section (day 7) of 9-10 sections from 2 independent experiments. Black asterisk indicates on large collection of blood in the lumen, arrowheads indicate on widespread mucosal collapse and ulceration and arrows indicates on edema in the sub mucosa.(F) A graphical summary of histological severity score of the indicated treatments.


( A - B ) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments . ( A ) Cytokine levels of IL - 6 , IL - 1β and INF - λ were determined by ELISA ( n = 4 - 7 ) . Results represent two or more independent experiments .

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(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (A) Cytokine levels of IL-6, IL-1β and INF-λ were determined by ELISA (n=4-7). Results represent two or more independent experiments.


( A - B ) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments . ( B ) mRNA expression levels of IL - 6 , IL - 12p35 and IL - 23p19 were assessed by quantitative RT - PCR and normalized to TBP ( n = 4 - 6 ) . Results represent two or more independent experiments .

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(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (B) mRNA expression levels of IL-6, IL-12p35 and IL-23p19 were assessed by quantitative RT-PCR and normalized to TBP (n=4-6). Results represent two or more independent experiments.


( A ) Flow cytometry analysis of colonic lamina propria CD11c - CD11b + LY6Chi MHCII - monocytes from steady - state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide , showing comparable monocytes infiltrates in to the colitic colon . Plots were pre gated on live CD45 + cells . Results are representative of three independent experiments ( n = 3 per group ) . ( B ) Graphical summary of monocytes presented as % out of CD11b + cells .

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(A) Flow cytometry analysis of colonic lamina propria CD11c- CD11b+LY6Chi MHCII-monocytes from steady-state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide, showing comparable monocytes infiltrates in to the colitic colon. Plots were pre gated on live CD45+ cells. Results are representative of three independent experiments (n=3 per group). (B) Graphical summary of monocytes presented as % out of CD11b + cells.


( B ) Weight loss of DSS challenged mice on day 6 .

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(B) Weight loss of DSS challenged mice on day 6.


( C ) Real - time PCR analysis showing mRNA expression levels of IL - 6 , IL - 12p35 and IL - 23p19 of FACS isolated monocytes collected from mice with the indicated treatments . Data are obtained from two to three independent experiments ( n = 6 - 7 mice pooled per group ) .

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(C) Real-time PCR analysis showing mRNA expression levels of IL-6, IL-12p35 and IL-23p19 of FACS isolated monocytes collected from mice with the indicated treatments. Data are obtained from two to three independent experiments (n=6-7 mice pooled per group).


RT - qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF - β for 16h normalized to MoDC . Data represent mean values ± SD of triplicates from two donors .

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RT-qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF-β for 16h normalized to MoDC. Data represent mean values ± SD of triplicates from two donors.


Subcellular localization analysis of S100A9 and S100A8 in MoLC , MoDC and MoDC - TGF - β . S100A9 mean fluorescence intensities ( MFI ) were normalized to MoLC and represented in the upper graph . The same was done for S100A9 cytoplasmic / nuclear MFI and shown in the lower graph . Scale bar : 5µm . Graphs show normalized percentages ± SD of data from four different donors for MoLC ( n = 4 ) and MoDC ( n = 4 ) and three different donors for TGF - β - treated MoDC ( n = 3 ) . When applicable , student t - test was performed to determine statistical significance * * P < 0 . 01 ) .

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Subcellular localization analysis of S100A9 and S100A8 in MoLC, MoDC and MoDC-TGF-β. S100A9 mean fluorescence intensities (MFI) were normalized to MoLC and represented in the upper graph. The same was done for S100A9 cytoplasmic/nuclear MFI and shown in the lower graph. Scale bar: 5µm. Graphs show normalized percentages ± SD of data from four different donors for MoLC (n=4) and MoDC (n=4) and three different donors for TGF-β-treated MoDC (n=3). When applicable, student t-test was performed to determine statistical significance **P<0.01).


MoLC from six different donors ( n = 6 ) were left untreated ( NT ) or incubated with LPS for 48h and analysed for S100A9 expression by RT - qPCR ( left graph ) . MoLC treated as before were then transduced with HIV - GFP vectors for 48h and GFP + cells were analysed by flow cytometry and data were graphically represented as fold relative to non - treated MoLC ( right graph ) . Black horizontal line correspond to the mean . When applicable , student t - test was performed to determine statistical significance ( * * * P < 0 . 001 ) .

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MoLC from six different donors (n=6) were left untreated (NT) or incubated with LPS for 48h and analysed for S100A9 expression by RT-qPCR (left graph). MoLC treated as before were then transduced with HIV-GFP vectors for 48h and GFP+ cells were analysed by flow cytometry and data were graphically represented as fold relative to non-treated MoLC (right graph). Black horizontal line correspond to the mean. When applicable, student t-test was performed to determine statistical significance (***P<0.001).


Cells transfected as above were pre - treated or not with nevirapine and transduced with HIV - GFP vectors for 48h . The percentage of GFP + MoLC was analysed by flow cytometry . Data represent means values ± SD from two different donors .

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Cells transfected as above were pre-treated or not with nevirapine and transduced with HIV-GFP vectors for 48h. The percentage of GFP+ MoLC was analysed by flow cytometry. Data represent means values ± SD from two different donors.


MoLC , dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis . Scale bar : 5µm .

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MoLC, dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis. Scale bar: 5µm.


Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV - Luc . When indicated cells were pre - treated with 10μM Nevirapine for 30 min before viral challenge . Luciferase activities measured in duplicates for each condition from two ( infection with 20 ng P24 ) or three ( infection with 200 ng P24 ) different skin samples were represented on a graph as means of relative luminescence units ( RLU ) ± SD .

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Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV-Luc. When indicated cells were pre-treated with 10μM Nevirapine for 30 min before viral challenge. Luciferase activities measured in duplicates for each condition from two (infection with 20 ng P24) or three (infection with 200 ng P24) different skin samples were represented on a graph as means of relative luminescence units (RLU) ± SD.


The experiment shown is representative of four donors for HIV - 1 - R5 ( n = 4 ) and three donors for HIV - 1 primary isolates ( n = 3 ) . Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV - 1 - R5 and HIV - 1 primary isolates . Nevirapine ( Nev ) ( 10μM ) was used as control when indicated and data were represented as means ± SD of HIV - Gag + cells from at least 2 ( for siS100A9 - MoLC infected by HIV - 1 primary isolates ) to 3 donors . When applicable , student t - test was performed to determine statistical significance ( * P < 0 . 1 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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The experiment shown is representative of four donors for HIV-1-R5 (n=4) and three donors for HIV-1 primary isolates (n=3). Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV-1-R5 and HIV-1 primary isolates. Nevirapine (Nev) (10μM) was used as control when indicated and data were represented as means ± SD of HIV-Gag+ cells from at least 2 (for siS100A9-MoLC infected by HIV-1 primary isolates) to 3 donors. When applicable, student t-test was performed to determine statistical significance (*P<0.1;**P<0.01;***P<0.001).


E2C - and S100A9 - expressing HEK293T cells were infected with the indicated MOI of HIV - GFP for 48h and GFP + cells were analysed by flow cytometry . Mean values ± SD from four independent experiments ( n = 4 ) were normalized to the E2C - HEK293T control and represented in graph .

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E2C- and S100A9-expressing HEK293T cells were infected with the indicated MOI of HIV-GFP for 48h and GFP+ cells were analysed by flow cytometry. Mean values ± SD from four independent experiments (n=4) were normalized to the E2C-HEK293T control and represented in graph.


The percentage of HIV - Gag p24 + cells from four independent experiments for CEM ( n = 4 ) and two for Jurkat ( n = 2 ) analysed by flow cytometry were graphically represented as means of HIV - Gag + cells ± SD . When applicable , student t - test was performed to determine statistical significance ( * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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The percentage of HIV-Gag p24+ cells from four independent experiments for CEM (n=4) and two for Jurkat (n=2) analysed by flow cytometry were graphically represented as means of HIV-Gag+ cells ± SD. When applicable, student t-test was performed to determine statistical significance (**P<0.01;***P<0.001).


E2C - and S100A9 - expressing CHO cells were challenged with indicated MOI of MMLV - YFP vectors for 24h or 48h . The percentages of YFP + CHO infected cells analysed by flow cytometry and data were represented on graphs .

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E2C- and S100A9-expressing CHO cells were challenged with indicated MOI of MMLV-YFP vectors for 24h or 48h. The percentages of YFP+ CHO infected cells analysed by flow cytometry and data were represented on graphs.


E2C - or S100A9 - expressing THP1 were left uninfected ( / ) or infected for 3 h with the indicated p24 amounts of HIV - 1 NL4 - 3 / Nef - IRES - Renilla viruses containing BlaM - VPR . Cells were processed for Blam - Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry . The mock condition corresponds to unstained E2C - expressing cells . Dot - plots from a representative experiment are shown .

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E2C- or S100A9-expressing THP1 were left uninfected (/) or infected for 3 h with the indicated p24 amounts of HIV-1 NL4-3/Nef-IRES-Renilla viruses containing BlaM-VPR. Cells were processed for Blam-Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry. The mock condition corresponds to unstained E2C-expressing cells. Dot-plots from a representative experiment are shown.


Schematic description of fate - of - capsid assay is displayed above . HEK293T expressing E2C or S100A9 were transduced with HIV - Luc ( 10ng p24 ) for 4 h . Cells were then subjected to the fate of capsid assay ( see methods ) in order to pull - down intact cores ( CA cores ) ( recovered in the pellet ) . Equal volumes from the soluble ( top fraction ) and pelleted ( down fraction ) materials were analyzed by western blotting in order to evaluate the presence of p24 ( CA ) , CypA , S100A9 and E2C proteins .

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Schematic description of fate-of-capsid assay is displayed above. HEK293T expressing E2C or S100A9 were transduced with HIV-Luc (10ng p24) for 4 h. Cells were then subjected to the fate of capsid assay (see methods) in order to pull-down intact cores (CA cores) (recovered in the pellet). Equal volumes from the soluble (top fraction) and pelleted (down fraction) materials were analyzed by western blotting in order to evaluate the presence of p24 (CA), CypA, S100A9 and E2C proteins.


Colorimetric HIV - 1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations . Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C - containing conditions and shown on graphs .

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Colorimetric HIV-1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations. Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C-containing conditions and shown on graphs.


Gene expression analyzed by quantitative PCR in brown adipocytes that were fed with or without 1mM asparagine as in ( A ) . n = 3 independent experiments . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed Student ' s t test .

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Gene expression analyzed by quantitative PCR in brown adipocytes that were fed with or without 1mM asparagine as in (A). n=3 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.


Preadipocytes bearing the shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine . Protein expression was analyzed on day 6 . n = 3 biological replicates .

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Preadipocytes bearing the shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine. Protein expression was analyzed on day 6. n=3 biological replicates.


Brown preadipocytes were infected by lentiviral shRNAs against Asns . Cells were differentiated in culture media with or without 1mM asparagine . Western blot was performed on day 6 .

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Brown preadipocytes were infected by lentiviral shRNAs against Asns. Cells were differentiated in culture media with or without 1mM asparagine. Western blot was performed on day 6.


UCP1 and PPARγ protein levels in BAT and inguinal WAT ( iWAT ) from mice in ( E ) .

UCP1 and PPARγ protein levels in BAT and inguinal WAT (iWAT) from mice in (E).


I , J . Mice supplemented with asparagine in drinking water for 3 weeks were subjected to the indirect calorimetry analysis at both basal ( I ) and CL316 , 243 - stimulated ( J ) states . n = 5 mice each group . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

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I,J. Mice supplemented with asparagine in drinking water for 3 weeks were subjected to the indirect calorimetry analysis at both basal (I) and CL316,243-stimulated (J) states. n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


Rectal temperature in mice of ( A ) during cold exposure . n = 5 mice each group .

Rectal temperature in mice of (A) during cold exposure. n=5 mice each group.


BAT and inguinal WAT ( iWAT ) gene expression analysis in mice of ( A ) .

BAT and inguinal WAT (iWAT) gene expression analysis in mice of (A).


Hematoxylin and eosin staining of BAT , inguinal WAT ( iWAT ) and epididymal WAT ( eWAT ) of mice in ( A ) .

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Hematoxylin and eosin staining of BAT, inguinal WAT (iWAT) and epididymal WAT (eWAT) of mice in (A).


Glucose tolerance test was performed after a high - fat diet feeding for 17 weeks in mice of ( A ) . n = 5 mice each group . Serum insulin levels in mice of ( A ) . n = 5 mice each group . Serum triglyceride levels in mice of ( A ) . n = 5 mice each group . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed Student ' s t test .

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Glucose tolerance test was performed after a high-fat diet feeding for 17 weeks in mice of (A). n=5 mice each group.   Serum insulin levels in mice of (A). n=5 mice each group.   Serum triglyceride levels in mice of (A). n=5 mice each group.   Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.


BAT and inguinal WAT ( iWAT ) UCP1 and PPARγ protein levels in mice of ( A ) .

BAT and inguinal WAT (iWAT) UCP1 and PPARγ protein levels in mice of (A).


Heat map of metabolite relative levels in brown fat cells treated with or without 1mM asparagine .

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Heat map of metabolite relative levels in brown fat cells treated with or without 1mM asparagine.


Immunoblot detection of the glucose transporters , glycolysis and TCA key enzymes in primary brown fat cell line Asparagine was supplemented to cells during differentiation .

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Immunoblot detection of the glucose transporters, glycolysis and TCA key enzymes in primary brown fat cell line Asparagine was supplemented to cells during differentiation.


Immunoblot detection of the glucose transporters , glycolysis and TCA key enzymes in immortalized brown fat cell line ( E ) , BAT from mice fed asparagine ( F ) and injected asparaginase ( G ) . Asparagine was supplemented to cells during differentiation .

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Immunoblot detection of the glucose transporters, glycolysis and TCA key enzymes in immortalized brown fat cell line (E), BAT from mice fed asparagine (F) and injected asparaginase (G). Asparagine was supplemented to cells during differentiation.


Intracellular abundance of 13C - isotopologue metabolites downstream of glycolysis in brown adipocytes . n = 5 independent experiments . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

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Intracellular abundance of 13C-isotopologue metabolites downstream of glycolysis in brown adipocytes. n=5 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


C , D . Quantification of glucose ( C ) and lactate ( D ) concentration from media . The media was collected after incubated with mature brown adipocytes for 48 h . n = 3 independent experiments in ( C ) , n = 6 independent experiments in ( D ) . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

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C,D. Quantification of glucose (C) and lactate (D) concentration from media. The media was collected after incubated with mature brown adipocytes for 48 h. n=3 independent experiments in (C), n=6 independent experiments in (D). Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


Quantification of intracellular amino acid levels in brown fat cells ( day 6 ) treated with or without 1mM asparagine . n = 4 independent experiments . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

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Quantification of intracellular amino acid levels in brown fat cells (day 6) treated with or without 1mM asparagine. n=4 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


B - D . mTORC1 pathway activity was analyzed by immunoblotting for the phosphorylation of 4E - BP1 and S6K in brown adipocytes ( B ) , BAT from mice fed asparagine ( C ) or asparaginase ( D ) . ( B ) Asparagine was added to cultured media from day 4 to day 6 .

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B-D. mTORC1 pathway activity was analyzed by immunoblotting for the phosphorylation of 4E-BP1 and S6K in brown adipocytes (B), BAT from mice fed asparagine (C) or asparaginase (D). (B) Asparagine was added to cultured media from day 4 to day 6.


Brown preadipocytes were induced to differentiation with or without 1mM asparagine . Rapamycin ( 75nM ) was added to cells for 48h . Then western blot detected protein expression on day 6 .

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Brown preadipocytes were induced to differentiation with or without 1mM asparagine. Rapamycin (75nM) was added to cells for 48h. Then western blot detected protein expression on day 6.


Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation . Protein expression analysis was performed on day 6 .

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Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation. Protein expression analysis was performed on day 6.


CSF p3 - Alcβ37 values in subjects who were categorized along the AD continuum ( n = 131 ) . Cut - off values are 359 . 6 pg / mL for Aβ42 ( A + indicates < 359 . 6 pg / mL ) , 30 . 6 pg / mL for p - tau ( T + indicates > 30 . 6 pg / mL ) , and 105 . 3 pg / mL for t - tau ( N + indicates > 105 . 3 pg / mL ) respectively . Statistical significance was determined by a one - way ANOVA followed by Tukey ' s multiple comparisons test ( means ± SEM ; A - : n = 36 , A + T - N - : n = 51 , A + T + N - : n = 30 , A + T + N + : n = 14 ) , and significant p - values ( p < 0 . 01 , p < 0 . 0001 ) are indicated on the graph .

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CSF p3-Alcβ37 values in subjects who were categorized along the AD continuum (n = 131). Cut-off values are 359.6 pg/mL for Aβ42 (A+ indicates <359.6 pg/mL), 30.6 pg/mL for p-tau (T+ indicates >30.6 pg/mL), and 105.3 pg/mL for t-tau (N+ indicates >105.3 pg/mL) respectively. Statistical significance was determined by a one-way ANOVA followed by Tukey's multiple comparisons test (means ± SEM; A-: n = 36, A+T-N-: n = 51, A+T+N-: n = 30, A+T+N+: n = 14), and significant p-values (p < 0.01, p < 0.0001) are indicated on the graph.


( C ) Effect of p3 - Alcβ37 and its partial peptides on the viability of mouse primary cultured neurons . Wild - type ( WT ) neurons ( div 15 - 20 ) were incubated for 24 h in the presence ( 10 μM ) or absence ( - ) of p3 - Alcβ1 - 11 , p3 - Alcβ9 - 19 , p3 - Alcβ20 - 37 , and p3 - Alcβ1 - 37 . Neuronal viability was evaluated with MTT assays and expressed relative to that of cells cultured in the absence of peptides ( assigned a value of 1 . 0 ) . Statistical analysis was performed using a one - way ANOVA , followed by Dunnett ' s multiple comparisons test ( means ± SEM ; n = 10 - 12 ) , and significant p - values ( p < 0 . 01 , p < 0 . 0001 ) are indicated on the graph .

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(C) Effect of p3-Alcβ37 and its partial peptides on the viability of mouse primary cultured neurons. Wild-type (WT) neurons (div 15-20) were incubated for 24 h in the presence (10 μM) or absence (-) of p3-Alcβ1-11, p3-Alcβ9-19, p3-Alcβ20-37, and p3-Alcβ1-37. Neuronal viability was evaluated with MTT assays and expressed relative to that of cells cultured in the absence of peptides (assigned a value of 1.0). Statistical analysis was performed using a one-way ANOVA, followed by Dunnett's multiple comparisons test (means ± SEM; n = 10-12), and significant p-values (p < 0.01, p < 0.0001) are indicated on the graph.


( D , E , F ) Effect of p3 - Alcβ on the neuronal toxicity of Aβ42 oligomers . WT neurons ( div 15 - 20 ) were incubated for 24 h in the presence ( 10 μM ) or absence ( - ) of p3 - Alcβ9 - 19 and p3 - Alcβ1 − 37 with ( + ) or without ( - ) Aβ42 oligomers ( Aβo , 2 . 5 μM ) . Neuronal viability was evaluated by MTT ( D ) , ATP generation ( E ) , and LDH release ( F ) assays and expressed relative to neurons cultured in the absence of p3 - Alcβ and Aβo ( assigned a value of 1 . 0 ) . Statistical significance was determined by Dunnett ' s multiple comparisons tests ( means ± SEM ; MTT : n = 36 , ATP : n = 40 , LDH : n = 60 ) . The significant p - values ( p < 0 . 05 , p < 0 . 01 , p < 0 . 001 , p < 0 . 0001 ) versus cells incubated in the absence ( - ) of p3 - Alcβ and Aβo are indicated in the graphs . The significant p - values ( # p < 0 . 05 , # p < 0 . 01 ) versus cells incubated in the presence ( + ) of Aβo and in the absence ( - ) of p3 - Alcβ ) are indicated in the graphs .

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(D, E, F) Effect of p3-Alcβ on the neuronal toxicity of Aβ42 oligomers. WT neurons (div 15-20) were incubated for 24 h in the presence (10 μM) or absence (-) of p3-Alcβ9-19 and p3-Alcβ1−37 with (+) or without (-) Aβ42 oligomers (Aβo, 2.5 μM). Neuronal viability was evaluated by MTT (D), ATP generation (E), and LDH release (F) assays and expressed relative to neurons cultured in the absence of p3-Alcβ and Aβo (assigned a value of 1.0). Statistical significance was determined by Dunnett's multiple comparisons tests (means ± SEM; MTT: n = 36, ATP: n = 40, LDH: n = 60). The significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) versus cells incubated in the absence (-) of p3-Alcβ and Aβo are indicated in the graphs. The significant p-values (#p < 0.05, #p < 0.01) versus cells incubated in the presence (+) of Aβo and in the absence (-) of p3-Alcβ) are indicated in the graphs.


( G ) Effect of Aβ42 oligomers ( Aβo ) and p3 - Alcβ peptides on the generation of reactive oxygen species ( ROS ) . WT neurons ( div 15 - 17 ) were incubated for 24 h with ( + ) or without ( - ) Aβo ( 2 . 5 μM ) and in the presence ( + ) or absence ( - ) of p3 - Alcβ ( 10 μM ) . ROS generation was assessed and expressed relative to that of neurons incubated in the absence ( - ) of Αβo , which was assigned a value of 1 . 0 . Statistical significance between samples with or without Aβo was evaluated with a Student ' s t - test ( means ± SEM ; n = 10 ) , and the significant p - value ( p < 0 . 05 ) is indicated on the graph .

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(G) Effect of Aβ42 oligomers (Aβo) and p3-Alcβ peptides on the generation of reactive oxygen species (ROS). WT neurons (div 15-17) were incubated for 24 h with (+) or without (-) Aβo (2.5 μM) and in the presence (+) or absence (-) of p3-Alcβ (10 μM). ROS generation was assessed and expressed relative to that of neurons incubated in the absence (-) of Αβo, which was assigned a value of 1.0. Statistical significance between samples with or without Aβo was evaluated with a Student's t-test (means ± SEM; n = 10), and the significant p-value (p < 0.05) is indicated on the graph.


( A , B ) Suppression of Ca2 + influx induced by Aβo in neurons treated with p3 - Alcβ9 - 19 ( A ) and p3 - Alcβ37 ( B ) . Mouse neurons ( div 11 - 13 ) pretreated with Fluo 4 - AM were stimulated at 5 min ( arrow ) with ( + ) or without ( - ) Aβo ( 5 . 2μM ) in the presence ( + ) or absence ( - ) of p3 - Alcβ ( 50 μM ) . The fluorescence intensity was recorded at the indicated time ( left ) and the fluorescence area intensity for 18 min ( 5 - 23 min at time points ) is shown ( right ) as the AUC expressed relative to that of cells cultured in the absence ( - ) of p3 - Alcβ and Aβo ( assigned a value of 1 . 0 ) .

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

(A, B) Suppression of Ca2+ influx induced by Aβo in neurons treated with p3-Alcβ9-19 (A) and p3-Alcβ37 (B). Mouse neurons (div 11-13) pretreated with Fluo 4-AM were stimulated at 5 min (arrow) with (+) or without (-) Aβo (5.2μM) in the presence (+) or absence (-) of p3-Alcβ (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 18 min (5-23 min at time points) is shown (right) as the AUC expressed relative to that of cells cultured in the absence (-) of p3-Alcβ and Aβo (assigned a value of 1.0).


( C , D ) Suppression of Ca2 + influx into neurons followed by Ca2 + administration in the presence of p3 - Alcβ9 - 19 ( C ) and p3 - Alcβ37 ( D ) . Mouse neurons ( div 11 - 13 ) pretreated with Fluo 4 - AM in calcium - depleted medium were administered Ca2 + ( 2 mM ) at 5 min ( arrow ) in the presence or absence of p3 - Alcβ9 - 19 ( C ) and p3 - Alcβ37 ( D ) . The fluorescence intensity was recorded at the indicated time ( left ) and the fluorescence area intensity for 7 . 5 or 9 min ( 5 to 12 . 5 min in panel C and 5 to 14 min in panel D at the indicated time points ) is shown ( right ) . Statistical significance was determined by one - way ANOVA with Tukey ' s multiple comparison test ( means ± SE ; n = 14 ( A ) , n = 15 ( B ) , n = 10 ( C ) ) . The significant p - values ( p < 0 . 05 , p < 0 . 01 , p < 0 . 001 , p < 0 . 0001 ) are indicated on the graphs . Statistical significance was determined with a Student ' s t - test ( means ± SEM ; n = 11 ( D ) ) , and the significant p - value ( p < 0 . 001 ) is indicated on the graph .

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

(C, D) Suppression of Ca2+ influx into neurons followed by Ca2+ administration in the presence of p3-Alcβ9-19 (C) and p3-Alcβ37 (D). Mouse neurons (div 11-13) pretreated with Fluo 4-AM in calcium-depleted medium were administered Ca2+ (2 mM) at 5 min (arrow) in the presence or absence of p3-Alcβ9-19 (C) and p3-Alcβ37 (D). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 7.5 or 9 min (5 to 12.5 min in panel C and 5 to 14 min in panel D at the indicated time points) is shown (right). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparison test (means ± SE; n = 14 (A), n = 15 (B), n = 10 (C)). The significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) are indicated on the graphs. Statistical significance was determined with a Student's t-test (means ± SEM; n = 11 (D)), and the significant p-value (p < 0.001) is indicated on the graph.


( A ) Suppression of Ca2 + influx induced by Aβ42 oligomers ( Aβo ) in neurons treated with the NMDA receptor antagonist D - AP5 . Neurons ( div 14 ) pretreated with Fluo 4 - AM were stimulated with or without Aβo ( 5 . 2 μM ) at 5 min ( arrow ) in the presence or absence of D - AP5 ( 50 μM ) . The fluorescence intensity was recorded at the indicated time ( left ) and the fluorescence area intensity for 24 min ( 5 - 29 min at the indicated time points ) is shown ( right ) as the AUC expressed relative to that of cells cultured in the absence of D - AP5 and Aβo ( assigned a value of 1 . 0 ) . Statistical significance was determined by one - way ANOVA with Tukey ' s multiple comparisons test ( means ± SE ; n = 4 ) , and significant p - values ( p < 0 . 05 , p < 0 . 01 ) are indicated on the graph .

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

(A) Suppression of Ca2+ influx induced by Aβ42 oligomers (Aβo) in neurons treated with the NMDA receptor antagonist D-AP5. Neurons (div 14) pretreated with Fluo 4-AM were stimulated with or without Aβo (5.2 μM) at 5 min (arrow) in the presence or absence of D-AP5 (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 24 min (5-29 min at the indicated time points) is shown (right) as the AUC expressed relative to that of cells cultured in the absence of D-AP5 and Aβo (assigned a value of 1.0). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test (means ± SE; n = 4), and significant p-values (p < 0.05, p < 0.01) are indicated on the graph.


( D ) Non - synergistic suppression of Ca2 + influx induced by Aβ42 oligomers ( Aβo ) in neurons by p3 - Alcβ and D - AP5 . Neurons ( div 14 ) pretreated with Fluo 4 - AM were stimulated with or without Aβo ( 5 . 2 μM ) at 5 min ( arrow ) in the presence ( + ) or absence ( - ) of p3 - Alcβ9 - 19 ( 50 μM ) and D - AP5 ( 50 μM ) . The fluorescence intensity was recorded at the indicated time ( left ) and the fluorescence area intensity for 23 min ( 5 - 28 min at the indicated time points ) is shown ( right ) as the AUC expressed relative to that of cells cultured in experiment No . 1 ( assigned a value of 1 . 0 ) . Statistical significance was determined with a one - way ANOVA with Tukey ' s multiple comparison test ( means ± SEM ; n = 12 ) , and significant p - values ( p < 0 . 05 , p < 0 . 01 , p < 0 . 001 , p < 0 . 0001 ) are indicated on the graph .

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

(D) Non-synergistic suppression of Ca2+ influx induced by Aβ42 oligomers (Aβo) in neurons by p3-Alcβ and D-AP5. Neurons (div 14) pretreated with Fluo 4-AM were stimulated with or without Aβo (5.2 μM) at 5 min (arrow) in the presence (+) or absence (-) of p3-Alcβ9-19 (50 μM) and D-AP5 (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 23 min (5-28 min at the indicated time points) is shown (right) as the AUC expressed relative to that of cells cultured in experiment No.1 (assigned a value of 1.0). Statistical significance was determined with a one-way ANOVA with Tukey's multiple comparison test (means ± SEM; n = 12), and significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) are indicated on the graph.


( E - G ) The SUVRs of groups administered p3 - Alcβ9 - 19 versus the vehicle - treated group . Statistical analysis was performed using one - way ANOVA , followed by Bonferroni correction for multiple comparisons ( means ± SEM ; n = 6 ) , and significant p - values ( p < 0 . 05 , p < 0 . 01 ) are indicated on the graphs .

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(E-G) The SUVRs of groups administered p3-Alcβ9-19 versus the vehicle-treated group. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni correction for multiple comparisons (means ± SEM; n = 6), and significant p-values (p < 0.05, p < 0.01) are indicated on the graphs.


( I , J ) The SUVRs of [ 18F ] BCPP - EF in the cortex ( I ) and hippocampus ( J ) are compared across the three groups . Statistical analysis was performed using one - way ANOVA , followed by Bonferroni correction for multiple comparisons ( means ± SEM ; n = 6 - 10 ) , and significant p - values ( p < 0 . 05 , p < 0 . 01 , p < 0 . 001 ) are indicated on the graphs .

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

(I, J) The SUVRs of [18F]BCPP-EF in the cortex (I) and hippocampus (J) are compared across the three groups. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni correction for multiple comparisons (means ± SEM; n = 6-10), and significant p-values (p < 0.05, p < 0.01, p < 0.001) are indicated on the graphs.


( K ) Improvement of the inverse SUVR correlation between [ 18F ] BCPP - EF and [ 11C ] DPA713 in AD mice following administration of p3 - Alcβ9 - 19 . AD mice with ( closed circle ) or without ( open circle ) subcutaneous injection of p3 - Alcβ9 - 19 peptide ( 1 mg / kg body weight ) are also analyzed with [ 11C ] DPA713 . A solid linear line shows a significant inverse correlation ( Pearson ' s correlation , r = 0 . 594 , p < 0 . 05 ) in AD mice , whereas a dotted linear line shows a positive correlation tendency in AD mice in response to p3 - Alcβ9 - 19 administration .

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(K) Improvement of the inverse SUVR correlation between [18F]BCPP-EF and [11C]DPA713 in AD mice following administration of p3-Alcβ9-19. AD mice with (closed circle) or without (open circle) subcutaneous injection of p3-Alcβ9-19 peptide (1 mg/kg body weight) are also analyzed with [11C]DPA713. A solid linear line shows a significant inverse correlation (Pearson's correlation, r = 0.594, p < 0.05) in AD mice, whereas a dotted linear line shows a positive correlation tendency in AD mice in response to p3-Alcβ9-19 administration.