I - J . Immunoblot for ubiquitin ( I ) and its quantification ( J ) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA ( sgCtrl ) or SERBP1 sgRNA ( sgSERBP1 ) along with INK128 treatment for 48 hours . At least four biological replicates are analyzed by unpaired t - test . Data are presented as the mean ± SD .

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I-J. Immunoblot for ubiquitin (I) and its quantification (J) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) along with INK128 treatment for 48 hours. At least four biological replicates are analyzed by unpaired t-test. Data are presented as the mean ± SD.


C , Comparison of wild - type colonies with phytochrome and hox - deletion strains in the presence of 0 . 12 mM menadion ( MD ) , 7 mM H2O2 or 2 mM tert - butyl hydroperoxide ( tBooH ) . The strains were incubated for 5 days at 28 ° C in the dark .

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C, Comparison of wild-type colonies with phytochrome and hox-deletion strains in the presence of 0.12 mM menadion (MD), 7 mM H2O2 or 2 mM tert-butyl hydroperoxide (tBooH). The strains were incubated for 5 days at 28°C in the dark.


E , Overlay of normalized ( at 705 nm ) difference spectra plus a difference spectrum of the photosensory domain in the presence of the bacterial HO

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E, Overlay of normalized (at 705 nm) difference spectra plus a difference spectrum of the photosensory domain in the presence of the bacterial HO BphO


B , HoxA - GFP and CitA - GFP ( ca . 170 amino acids from the N - terminus of citrate synthase ) were expressed over night at 30 ° C in minimal medium with 2 % threonine and 0 . 2 % glucose . Mitochondria were incubated with 100 μg / ml proteinase K ( PK ) for 20 min on ice . Crude extract ( CE ) , supernatant 1 ( S1 ) containing mitochondria , pellet 1 ( P1 ) , supernatant 2 ( S2 ) and pellet 2 ( P2 ) with mitochondria were analyzed by Western Blot using anti GFP antibodies . CitA - GFPc = cytoplasmic form ; CitA - GFPi = imported version .

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B, HoxA-GFP and CitA-GFP (ca. 170 amino acids from the N-terminus of citrate synthase) were expressed over night at 30°C in minimal medium with 2% threonine and 0.2% glucose. Mitochondria were incubated with 100 μg/ml proteinase K (PK) for 20 min on ice. Crude extract (CE), supernatant 1 (S1) containing mitochondria, pellet 1 (P1), supernatant 2 (S2) and pellet 2 (P2) with mitochondria were analyzed by Western Blot using anti GFP antibodies. CitA-GFPc = cytoplasmic form; CitA-GFPi = imported version.


C , D Role of the C - terminal anchor in HoxA . Microscopy was done as in A using a fluorescent microscope without the Airyscan technology . C , Expression of GFP - HoxA lacking the C - terminal anchor ( ∆ CTA ) in A . alternata . D , Expression of GFP fused to the CTA motif .

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C, D Role of the C-terminal anchor in HoxA. Microscopy was done as in A using a fluorescent microscope without the Airyscan technology. C, Expression of GFP-HoxA lacking the C-terminal anchor (∆CTA) in A. alternata. D, Expression of GFP fused to the CTA motif.


A , BiFC ( split - YFP ) analysis of HOs and phytochrome . Strains ( SChS29 - 33 ) were grown over night at 28 ° C minimal medium with 2 % threonine and 0 . 2 % glucose . MitoTracker Red CMOXRos was used for mitochondrial staining . The mitotracker signal was observed in the RFP and the split - YFP signal in the YFP channel of the fluorescent microscope . Pictures in both channels were overlaid ( merge ) . The left pictures show the same hyphae in differential interference contrast ( DIC ) . The scale bar represents 10 µm .

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A, BiFC (split-YFP) analysis of HOs and phytochrome. Strains (SChS29-33) were grown over night at 28°C minimal medium with 2% threonine and 0.2% glucose. MitoTracker Red CMOXRos was used for mitochondrial staining. The mitotracker signal was observed in the RFP and the split-YFP signal in the YFP channel of the fluorescent microscope. Pictures in both channels were overlaid (merge). The left pictures show the same hyphae in differential interference contrast (DIC). The scale bar represents 10 µm.


C , Co - expression of the HOXs along with the photosensory domain of A . nidulans FphA ( PGP ) over night at 20 ° C . PGP was purified via the Strep - tag system and the eluate concentrated ( upper panel ) . The lanes were loaded as follows : negative control before induction ( - ) , 20 h after induction ( 20 h ) crude extract ( CE ) , supernatant ( S ) , pellet ( P ) , flow through ( F ) and eluate ( E ) . The fractions were analyzed by Western Blot using the Anti - His antibody ( lower panel ) .

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C, Co-expression of the HOXs along with the photosensory domain of A. nidulans FphA (PGP) over night at 20°C. PGP was purified via the Strep-tag system and the eluate concentrated (upper panel). The lanes were loaded as follows: negative control before induction (-), 20 h after induction (20 h) crude extract (CE), supernatant (S), pellet (P), flow through (F) and eluate (E). The fractions were analyzed by Western Blot using the Anti-His antibody (lower panel).


A , B , Biolayer interferometry analysis with Apo - PGP A and holo - PGP B . Proteins were immobilized at a streptavidin sensor tip . Free binding sites were blocked with biotin . Association and dissociated kinetics were recorded at the indicated HoxA concentrations . Kd values were calculated with the manufacturers software using a global fit for both

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A, B, Biolayer interferometry analysis with Apo-PGP A and holo-PGP B. Proteins were immobilized at a streptavidin sensor tip. Free binding sites were blocked with biotin. Association and dissociated kinetics were recorded at the indicated HoxA concentrations. Kd values were calculated with the manufacturers software using a global fit for both experiment


( B ) BW25113 ( ycbB , relA ' ) and its derivatives obtained by individual deletion of endopeptidase genes . BW25113 ( ycbB , relA ' ) is an abbreviated name for BW25113 ΔrelA pKT2 ( ycbB ) pKT8 ( relA ' ) .

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(B) BW25113(ycbB, relA') and its derivatives obtained by individual deletion of endopeptidase genes. BW25113(ycbB, relA') is an abbreviated name for BW25113 ΔrelA pKT2(ycbB) pKT8(relA').


Growth of BW25113 ( ycbB , relA ' ) , BW25113 ( ycbB , relA ' ) ΔmepM , and its derivatives harboring plasmids encoding MepM , MepM H393A , and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg / ml ( + ceftriaxone ) or in the absence of the drug ( - ceftriaxone ) in BHI agar plates supplemented with 40 µM IPTG and 1 % L - arabinose for induction of ycbB and relA ' , respectively . The genes encoding MepM , MepM H393A , and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter , which is inducible by 2 , 4 - diacetylphloroglucinol ( DAPG ) . Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer .

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Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM H393A, and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) in BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. The genes encoding MepM, MepM H393A, and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter, which is inducible by 2,4-diacetylphloroglucinol (DAPG). Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer.


Growth of BW25113 ( ycbB , relA ' ) , BW25113 ( ycbB , relA ' ) ΔmepM , and its derivatives harboring plasmids encoding MepM ,

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Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM


The mepM gene was expressed under the control of TIS1 or TIS2 ( translation initiation ) and of PrhaBAD ( the L - rhamnose - inducible promoter of vector pHV30 ) . Growth of BW25113 ( ycbB , relA ' ) ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg / ml , IPTG 40 µM ( induction of ycbB ) and L - arabinose 1 % ( induction of relA ' ) . ( A ) For TIS2 , growth around paper disks containing 1 or 2 mg of L - rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance . In contrast , a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer . ( B ) The experiment was repeated with bacteria pre - exposed to L - rhamnose that were harvested at the vicinity of the disk containing 2 mg of L - rhamnose . Expression of ceftriaxone resistance remained dependent on the presence of L - rhamnose indicating that the requirement for MepM is not transient . The diameter of the growth zones is larger in panel ( B ) than in panel ( A ) as expected from the fact that bacteria in the inoculum used in ( B ) had been grown in the presence of the inducer and already contained MepM . In ( A ) growth is only possible after diffusion of L - rhamnose in the medium prior to the action of ceftriaxone . At a distance from the disk , diffusion was not sufficiently rapid to observe resistance .

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The mepM gene was expressed under the control of TIS1 or TIS2 (translation initiation) and of PrhaBAD (the L-rhamnose-inducible promoter of vector pHV30). Growth of BW25113(ycbB, relA') ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg/ml, IPTG 40 µM (induction of ycbB) and L-arabinose 1% (induction of relA'). (A) For TIS2, growth around paper disks containing 1 or 2 mg of L-rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance. In contrast, a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer. (B) The experiment was repeated with bacteria pre-exposed to L-rhamnose that were harvested at the vicinity of the disk containing 2 mg of L-rhamnose. Expression of ceftriaxone resistance remained dependent on the presence of L-rhamnose indicating that the requirement for MepM is not transient. The diameter of the growth zones is larger in panel (B) than in panel (A) as expected from the fact that bacteria in the inoculum used in (B) had been grown in the presence of the inducer and already contained MepM. In (A) growth is only possible after diffusion of L-rhamnose in the medium prior to the action of ceftriaxone. At a distance from the disk, diffusion was not sufficiently rapid to observe resistance.


( A ) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme ( upper panel ) or by lysozyme and 5 µM MepM ( lower panel ) . Absorbance was monitored at 205 nm ( mAU , milli - absorbance unit ) .

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(A) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme (upper panel) or by lysozyme and 5 µM MepM (lower panel). Absorbance was monitored at 205 nm (mAU, milli-absorbance unit).


Functional complementation of the mepM deletion in BW25113 ( ycbB , relA ' ) ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal . Induction of endopeptidase ( ED ) genes was performed with 0 . 2 % L - rhamnose in the presence or absence of 8 µg / ml ceftriaxone . BHI agar plates contained 40 µM IPTG and 1 % L - arabinose for induction of ycbB and relA ' , respectively .

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Functional complementation of the mepM deletion in BW25113(ycbB, relA') ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal. Induction of endopeptidase (ED) genes was performed with 0.2% L-rhamnose in the presence or absence of 8 µg/ml ceftriaxone. BHI agar plates contained 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively.


( B ) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53 ( mepM ) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter . In this assay , functional replacement of MepM is detected based on growth in media containing 0 . 2 % or 1 % L - arabinose for expression of the endopeptidase gene carried by vector pHV7 , while by - passing the requirement for induction of the mepM copy of pHV53 by L - rhamnose . Complementation was observed with both concentrations of inducer for mepH , mepS , and pbpG . Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L - arabinose ( 1 % ) was lethal . The right panel presents the growth control performed in the presence of 0 . 2 % L - rhamnose for induction of the mepM copy carried by pHV53 .

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(B) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53(mepM) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter. In this assay, functional replacement of MepM is detected based on growth in media containing 0.2% or 1% L-arabinose for expression of the endopeptidase gene carried by vector pHV7, while by-passing the requirement for induction of the mepM copy of pHV53 by L-rhamnose. Complementation was observed with both concentrations of inducer for mepH, mepS, and pbpG. Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L-arabinose (1%) was lethal. The right panel presents the growth control performed in the presence of 0.2% L-rhamnose for induction of the mepM copy carried by pHV53.


( B ) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter . Induction of ycbB and of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose and 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

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(B) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


( A ) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with the pHV30 vector or recombinant plasmids encoding mepA , mepK , mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter . ( B ) The complementation assay was repeated for BW25113 Δ7EDs pHV63 ( ycbB ) , which was obtained by deletion of all chromosomal endopeptidase genes except mepM . Induction of ycbB and of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose and 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

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(A) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding mepA, mepK, mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter. (B) The complementation assay was repeated for BW25113 Δ7EDs pHV63(ycbB), which was obtained by deletion of all chromosomal endopeptidase genes except mepM. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


( A ) Growth curves of ( i ) BW25113 ( ycbB , relA ' ) and its ΔsltY derivative ; and ( ii ) strain M1 and its derivatives ( M1 . 1 to 1 . 4 ) harboring mutations in sltY . The growth medium contained ( i ) 40 µM IPTG , 1 % L - arabinose , and 8 µg / ml ceftriaxone for BW25113 ( ycbB , relA ' ) and its ΔsltY derivative or ( ii ) 50 µM IPTG and 16 µg / ml ampicillin for M1 and its derivatives .

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(A) Growth curves of (i) BW25113(ycbB, relA') and its ΔsltY derivative; and (ii) strain M1 and its derivatives (M1.1 to 1.4) harboring mutations in sltY. The growth medium contained (i) 40 µM IPTG, 1% L-arabinose, and 8 µg/ml ceftriaxone for BW25113(ycbB, relA') and its ΔsltY derivative or (ii) 50 µM IPTG and 16 µg/ml ampicillin for M1 and its derivatives.


A . Intracellular survival of GAS strains 188 , 188SLO - ( SLO - ) , and 188NADase - ( NADase - ) . Intracellular CFU were quantified at 2 h , 4 h , 6 h , 8 h , and 24 h , and intracellular survival was calculated as the percent viable CFU compared to 2 h .

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A. Intracellular survival of GAS strains 188, 188SLO- (SLO-), and 188NADase- (NADase-). Intracellular CFU were quantified at 2 h, 4 h, 6 h, 8 h, and 24 h, and intracellular survival was calculated as the percent viable CFU compared to 2 h.


B . Intracellular survival of GAS strain 188SLO - that was partially complemented from plasmid piSLO ( SLO - ( piSLO ) ) , strain 188 and 188SLO - that contained the empty complementation vector , 188 ( pSIV4 ) and SLO - ( pSIV4 ) , respectively .

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B. Intracellular survival of GAS strain 188SLO- that was partially complemented from plasmid piSLO (SLO-(piSLO)), strain 188 and 188SLO- that contained the empty complementation vector, 188(pSIV4) and SLO-(pSIV4), respectively.


C . Intracellular survival of GAS strain JRS4 , JRS4SLO - ( SLO - ) , and JRS4NADase - ( NADase - ) . Experiments were performed in triplicate and values represent the mean of three independent experiments ± SD . * , P < 0 . 001 .

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C. Intracellular survival of GAS strain JRS4, JRS4SLO- (SLO-), and JRS4NADase- (NADase-). Experiments were performed in triplicate and values represent the mean of three independent experiments ± SD. *, P<0.001.


A . Confocal microscopy of the association between EGFP - LC3 and GAS strain 188 , 188SLO - ( SLO - ) , 188SLS - ( SLS - ) , or 188SLO - SLS - ( SLO - SLS - ) . Immunofluorescent staining distinguished intracellular ( Alexa - 568 , red ) from extracellular ( Alexa - 568 and Alexa - 660 , red and blue , respectively ) GAS . Scale bar = 10 µm . The percent of intracellular GAS that were associated with EGFP - LC3 at 3 h post - infection is shown for each strain .

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A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain.


B . Electron microscopy of GAS strains 188 , 188SLO - , and 188SLO - SLS - at 3 h post - infection . Arrowheads indicate the presence of double or multiple membranes ( 188 and 188SLO - ) or single membranes ( 188SLO - SLS - ) partially or completely surrounding bacteria . Vacuolar compartments associated with 188 and 188SLO - also contain cytosolic material .

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B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material.


C . Intracellular survival of GAS strains 188 , 188SLO - , 188SLS - , and 188SLO - SLS - . * , P < 0 . 05 .

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C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05.


D . Intracellular survival of 188 or 188SLO - in the presence or absence of Beclin1 knockdown .

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D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.


A . Intracellular survival of GAS strains 188 , 188NADase - , and 188NADase ( G330D ) . Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20 - to 40 - fold reduction in intracellular survival compared to parent strain 188 . * , P < 0 . 001 .

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A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001.


and activity measurements ( C ) of culture supernatants from GAS strains 188 , 188NADase - , and 188NADase ( G330D ) .

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and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).


A . Confocal microscopy demonstrating the association between ubiquitin ( green ) and intracellular GAS ( red ) at 30 min post - infection in keratinocytes infected with GAS strain 188 , 188SLO - , 188NADase - , or 188SLO ( Y255A ) . Extracellular GAS were stained blue and red for identification as described in Figure 2 . Scale bar = 10 µm . The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain , based on quantification of at least 100 bacteria in three independent experiments .

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A. Confocal microscopy demonstrating the association between ubiquitin (green) and intracellular GAS (red) at 30 min post-infection in keratinocytes infected with GAS strain 188, 188SLO-, 188NADase-, or 188SLO(Y255A). Extracellular GAS were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments.


B . Hemolytic activity of culture supernatants of GAS strain 188 , 188SLO - , and 188SLO ( Y255A ) . The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane , demonstrated by the failure of culture supernatants from 188SLO ( Y255A ) to lyse erythrocytes despite producing wild - type amounts of the variant SLO protein . Inset , corresponding Western blot for SLO in culture supernatants from these strains .

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B. Hemolytic activity of culture supernatants of GAS strain 188, 188SLO-, and 188SLO(Y255A). The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane, demonstrated by the failure of culture supernatants from 188SLO(Y255A) to lyse erythrocytes despite producing wild-type amounts of the variant SLO protein. Inset, corresponding Western blot for SLO in culture supernatants from these strains.


. C . NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771 , 771SLO - , 771SLO ( Y255A ) , or 771NADase - . Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO ( Y255A ) , but reduced in 771SLO - .

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. C. NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771, 771SLO-, 771SLO(Y255A), or 771NADase-. Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO(Y255A), but reduced in 771SLO-.


D . The intracellular survival of 188SLO ( Y255A ) was significantly lower than that of its parental strain , 188 , and similar to that of 188SLO - . * , P < 0 . 01 .

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D. The intracellular survival of 188SLO(Y255A) was significantly lower than that of its parental strain, 188, and similar to that of 188SLO-. *, P<0.01.


E . Confocal microscopy demonstrating the association between galectin 8 ( green ) and intracellular GAS ( red ) in keratinocytes infected for 30 min with GAS strain 188 , 188SLO - , or 188 SLO - SLS - . Extracellular GAS ( GAS ( out ) ) were stained blue and red for identification as described in Figure 2 . Scale bar = 10 µm . The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain , based on quantification of at least 100 bacteria in three independent experiments . 188SLO - SLS - was never associated with galectin 8 ( 0 % ) .

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E. Confocal microscopy demonstrating the association between galectin 8 (green) and intracellular GAS (red) in keratinocytes infected for 30 min with GAS strain 188, 188SLO-, or 188 SLO-SLS-. Extracellular GAS (GAS(out)) were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments. 188SLO-SLS- was never associated with galectin 8 (0%).


SLO and NADase inhibit lysosomal fusion to GAS - containing autophagosomes in oropharyngeal keratinocytes . A . Confocal microscopy of keratinocytes infected with GAS strain 188 , 188SLO - , or 188NADase - demonstrating the association of the lysosomal marker LAMP - 1 ( red ) with GAS ( blue ) contained within EGFP - LC3 ( green ) - positive compartments at 1 h , 3 h , and 6 h post - infection . Scale bar = 10 µm . B . Quantification of the percent of GAS within EGFP - LC3 - positive compartments that are co - localized with LAMP - 1 at 1 h , 3 h , and 6 h . Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment .

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SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.


( B ) Representative immunoblot of Nrxn1β - Fc variants harvested from HEK293 cell media and subjected to heparinase treatment ( ' Heps ' ) . Samples were analyzed by reducing SDS / PAGE and immunoblotting with antibodies against the Fc - tag and the 3G10 epitope ( to detect the HS stub which reveal HS after heparinase digestion ) . ( C ) Quantification of 3G10 ( reflecting HS ) signal normalized to that of Fc ( Nrxn1 ) . Bar graph shows means ± SEM of 3 independent experiments .

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(B) Representative immunoblot of Nrxn1β-Fc variants harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against the Fc-tag and the 3G10 epitope (to detect the HS stub which reveal HS after heparinase digestion). (C) Quantification of 3G10 (reflecting HS) signal normalized to that of Fc (Nrxn1). Bar graph shows means ±SEM of 3 independent experiments.


HEK293 cells : ( B ) Representative immunoblots ( of three independent experiments ) with antibodies against HA , the 3G10 epitope ( for the HS stub ) and V5 .

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HEK293 cells: (B) Representative immunoblots (of three independent experiments) with antibodies against HA, the 3G10 epitope (for the HS stub) and V5.


HEK293 cells : ( D ) Representative immunoblots ( of three independent experiments ) analyzed as described for ( B ) .

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HEK293 cells: (D) Representative immunoblots (of three independent experiments) analyzed as described for (B).


( F - H ) Primary cortical neurons : Cortical neurons were infected with control lentivirus ( empty vector ) or lentivirus expressing FLAG - tagged CA10 , and cell lysates subjected to immunoprecipitation for neurexin and heparinase treatment . ( F ) Samples were analyzed by immunoblotting using antibodies against neurexins , 3G10 ( for the HS stub ) and FLAG ( for CA10 ) . Stars indicate non - neurexin bands ( see results ) . ( G ) Quantification of low molecular weight neurexin ( LMW ; white arrowhead ) in relation to total neurexin amounts [ upper band ( line ) + LMW ] . Data shown as mean ± SEM ( n = 3 independent experiments ) ; * , p < 0 . 05 by 2 - way ANOVA and Holm - Sidak tests . ( H ) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band . Data shown as mean ± SEM of biological replicates . * , p < 0 . 05 by 2 - way ANOVA and Holm - Sidak tests ( n = 3 ) .

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(F-H) Primary cortical neurons: Cortical neurons were infected with control lentivirus (empty vector) or lentivirus expressing FLAG-tagged CA10, and cell lysates subjected to immunoprecipitation for neurexin and heparinase treatment. (F) Samples were analyzed by immunoblotting using antibodies against neurexins, 3G10 (for the HS stub) and FLAG (for CA10). Stars indicate non-neurexin bands (see results). (G) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (H) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests (n=3).


( I - K ) Mouse brains : Neurexins and CA10 were immunoprecipitated from total mouse brain lysates and subject to heparinase treatment . ( I ) Samples were analyzed by immunoblotting using antibodies against neurexins , the 3G10 epitope ( for the HS stub ) and CA10 . Stars indicate non - neurexin bands ( see results ) . ( J ) Quantification of low molecular weight neurexin ( LMW ; white arrowhead ) in relation to total neurexin amounts [ upper band ( line ) + LMW ] . Data shown as mean ± SEM ( n = 3 independent experiments ) ; * , p < 0 . 05 by 2 - way ANOVA and Holm - Sidak tests . ( K ) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band . Data shown as mean ± SEM of 3 biological replicates . * , p < 0 . 05 by 2 - way ANOVA and Holm - Sidak tests .

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(I-K) Mouse brains: Neurexins and CA10 were immunoprecipitated from total mouse brain lysates and subject to heparinase treatment. (I) Samples were analyzed by immunoblotting using antibodies against neurexins, the 3G10 epitope (for the HS stub) and CA10. Stars indicate non-neurexin bands (see results). (J) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (K) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of 3 biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests.


( B ) Representative immunoblot of the indicated proteins harvested from HEK293 cell media and subjected to heparinase treatment ( ' Heps ' ) . Samples were analyzed by reducing SDS / PAGE and immunoblotting with antibodies against Fc ( for Nrxn1β ) , the 3G10 epitope ( for the HS stub ) and V5 ( for CA10 ) . Input samples were analyzed separately for CA10 to ensure equal expression ( lower panel ) . ( C ) Quantification of the 3G10 epitope signal , normalized to Fc ( Nrxn1β ) . Data shown are mean ± SEM of 3 independent replicates . * , p < 0 . 05 ; by Mann - Whitney tests comparing each Nrxn1 mutant with and without CA10 ( n = 3 ) . No significance difference was observed for S & T > G ( p = 0 . 1143 ) , GIGG ( p = 0 . 9714 ) or DIRR ( p = 0 . 4857 ) variants .

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(B) Representative immunoblot of the indicated proteins harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against Fc (for Nrxn1β), the 3G10 epitope (for the HS stub) and V5 (for CA10). Input samples were analyzed separately for CA10 to ensure equal expression (lower panel). (C) Quantification of the 3G10 epitope signal, normalized to Fc (Nrxn1β). Data shown are mean ±SEM of 3 independent replicates. *, p< 0.05; by Mann-Whitney tests comparing each Nrxn1 mutant with and without CA10 (n=3). No significance difference was observed for S&T>G (p=0.1143), GIGG (p=0.9714) or DIRR (p=0.4857) variants.


( E ) representative immunoblots analyzed using antibodies against the Fc - tag ( for Nrxn1β - Fc ) the 3G10 epitope ( for the HS stub ) and V5 ( CA10 - V5 ) , as indicated . CA10 in input samples is shown for demonstration of equal expression . ( F , Quantification of the 3G10 epitope signal normalized to Fc ( for Nrxn1β - Fc ) signals , Data shown are mean ± SEM of 3 biological replicates . * , p < 0 . 05 ; * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests .

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(E) representative immunoblots analyzed using antibodies against the Fc-tag (for Nrxn1β-Fc) the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. (F, Quantification of the 3G10 epitope signal normalized to Fc (for Nrxn1β-Fc) signals, Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.


( G ) show representative immunoblots analyzed using antibodies against the HA tag ( Nrxn1α - HA ) , the 3G10 epitope ( for the HS stub ) and V5 ( CA10 - V5 ) , as indicated . CA10 in input samples is shown for demonstration of equal expression . H ) Quantification of the 3G10 epitope signal normalized to HA ( for Nrxn1α - HA ) signals , respectively . Data shown are mean ± SEM of 3 biological replicates . * , p < 0 . 05 ; * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests .

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(G) show representative immunoblots analyzed using antibodies against the HA tag (Nrxn1α-HA), the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. H) Quantification of the 3G10 epitope signal normalized to HA (for Nrxn1α-HA) signals, respectively. Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.


( D ) Comparison of 1H - 13C HSQC spectra of Nrxn1 stalk ( red ) and Nrxn1 stalk / CA10 ( black ) at a 2 : 1 molar ratio ( 50 mM phosphate buffer , pH 7 . 4 , 0 . 5 mM d10 - DTT , 10 % D2O ) . Nrxn1 residues with stronger chemical shift changes are indicated blue boxes . Asterisk ( * ) indicates an impurity from glycerol .

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(D) Comparison of 1H-13C HSQC spectra of Nrxn1 stalk (red) and Nrxn1 stalk/CA10 (black) at a 2:1 molar ratio (50 mM phosphate buffer, pH 7.4, 0.5 mM d10-DTT, 10% D2O). Nrxn1 residues with stronger chemical shift changes are indicated blue boxes. Asterisk (*) indicates an impurity from glycerol.


( B - C ) Extracted ion chromatograms ( EIC ) of DDILVASAECPSDDE peptides and its glycosylated forms , from Nrxn1β expressed in HEK293 cells .

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(B-C) Extracted ion chromatograms (EIC) of DDILVASAECPSDDE peptides and its glycosylated forms, from Nrxn1β expressed in HEK293 cells.


( D ) Quantification , expressed as % of total occupancy of each glycosylation form , upon control and CA10 expression conditions . Bar graph shows means ± SEM of 4 biological replicates ; * , p < 0 . 05 by Mann - Whitney tests comparing the occupancy of each glycosylation with and without CA10 ( n = 4 biological replicates ) .

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(D) Quantification, expressed as % of total occupancy of each glycosylation form, upon control and CA10 expression conditions. Bar graph shows means ±SEM of 4 biological replicates; *, p<0.05 by Mann-Whitney tests comparing the occupancy of each glycosylation with and without CA10 (n=4 biological replicates).


( A ) Immunoblot α - showing neurexin isoform distribution in cortex , hippocampus , midbrain and cerebellum dissected from 8 - week - old mice . ( B ) Quantification of lower molecular weight neurexin ( LMW ; white arrowhead ) in relation to total levels of α - neurexins [ upper band ( line ) + LMW ] . Bar graphs show mean ± SEM of 3 independent replicates * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests , comparing to levels in cerebellum .

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(A) Immunoblot α-showing neurexin isoform distribution in cortex, hippocampus , midbrain and cerebellum dissected from 8-week-old mice. (B) Quantification of lower molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ± SEM of 3 independent replicates***, p<0.001 by 2-way ANOVA and Holm-Sidak tests, comparing to levels in cerebellum.


( C ) α - Neurexin immunoprecipitated from forebrain ( Fb ) and cerebellum ( Cb ) using an anti - pan - neurexin antibody was heparinase - treated ( ' Heps ' ) and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal ( to detect the HS stub ) . Stars indicate non - neurexin bands ( see results ) . ( D ) Quantification of low molecular weight neurexin ( LMW ; white arrowhead ) in relation to total levels of α - neurexins [ upper band ( line ) + LMW ] . Bar graphs show mean ± SEMof 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests comparing forebrain and cerebellum . ( E ) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band . Bar graphs show mean ± SEM of 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests comparing forebrain and cerebellum .

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(C) α-Neurexin immunoprecipitated from forebrain (Fb) and cerebellum (Cb) using an anti-pan-neurexin antibody was heparinase-treated ('Heps') and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal (to detect the HS stub). Stars indicate non-neurexin bands (see results). (D) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ±SEMof 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum. (E) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Bar graphs show mean ±SEM of 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum.


( A - B ) Cell surface binding to endogenous neurexins . ( A ) Recombinant Fc - tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control ( empty vector ) by lentiviral transduction . Surface - bound protein was quantified as the Fc signal of and normalized to a staining for endogenous neurexin ( n = 4 cultures ; three fields imaged and averaged per well ) . Scatchard analysis for LRRTM2 revealed an apparent Kd of 29 . 5 nM and a Bmax of 0 . 49 nM for control and Kd 35 . 3 nM , Bmax 0 . 28 nM upon expression of CA10 ; significant ( p = 0 . 0006 ) by non - linear regression . Data shown as mean ± SEM of biological replicates ( n = 4 ) . ( B ) Increasing amounts of recombinant , Fc - tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control ( empty vector ) by lentiviral transduction , and analyzed as in ( A ) . The apparent Kd for Nlgn1 was 186 nM and Bmax 1 . 65 nM for control and Kd 223 nM , Bmax 1 . 49 nM when CA10 was overexpressed . The difference was not significant ( p = 0 . 20 ) by non - linear regression . Data shown as mean ± SEM of biological replicates ( n = 4 ) .

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

(A-B) Cell surface binding to endogenous neurexins. (A) Recombinant Fc-tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction. Surface-bound protein was quantified as the Fc signal of and normalized to a staining for endogenous neurexin (n=4 cultures; three fields imaged and averaged per well). Scatchard analysis for LRRTM2 revealed an apparent Kd of 29.5 nM and a Bmax of 0.49 nM for control and Kd 35.3 nM, Bmax 0.28 nM upon expression of CA10; significant (p=0.0006) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4). (B) Increasing amounts of recombinant, Fc-tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction, and analyzed as in (A). The apparent Kd for Nlgn1 was 186 nM and Bmax 1.65 nM for control and Kd 223 nM, Bmax 1.49 nM when CA10 was overexpressed. The difference was not significant (p=0.20) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4).


( C - D ) Artificial synapse formation assays . ( C ) Confocal images of hippocampal neurons transduced to express CA10 - FLAG or control ( empty vector ) and co - cultured with HEK293 cells expressing GFP , Nlgn1 - YFP , LRRTM2 - YFP or TrkC - YFP . Cells were stained with antibodies against the synaptic marker SV2 ( red ) and somatodendritic MAP2 ( blue ) ; GFP / YFP is shown in green . Scale bar 20 μm . ( D ) Quantifications of SV2 signal overlapping GFP / YFP - positive cells . Data shown as means ± SEM with the n , reflecting number of independent cover slips analyzed , indicated in each bar with the total number of cells imaged in parenthesis . Comparisons were made by one - way ANOVA with Holm - Sidak ' s posthoc test for multiple comparisons between the indicated groups ( GFP condition was omitted from analysis due to unequal variance ; * * * , p < 0 . 001 .

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(C-D) Artificial synapse formation assays. (C) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured with HEK293 cells expressing GFP, Nlgn1-YFP, LRRTM2-YFP or TrkC-YFP. Cells were stained with antibodies against the synaptic marker SV2 (red) and somatodendritic MAP2 (blue); GFP/YFP is shown in green. Scale bar 20 μm. (D) Quantifications of SV2 signal overlapping GFP/YFP-positive cells. Data shown as means ±SEM with the n, reflecting number of independent cover slips analyzed, indicated in each bar with the total number of cells imaged in parenthesis. Comparisons were made by one-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups (GFP condition was omitted from analysis due to unequal variance; ***, p<0.001.


( E - F ) Analysis of cerebellin ( Cbln ) - induced artificial synapse formation . ( E ) Confocal images of hippocampal neurons transduced to express CA10 - FLAG or control ( empty vector ) and co - cultured exposed to anti - V5 - coupled beads pre - incubated with recombinant V5 - tagged Cbln1 or empty anti - V5 beads . Cells were stained with antibodies against the synaptic marker synapsin ( green ) , somatodendritic MAP2 ( blue ) ; beads are colored red and indicated by arrowheads . Scale bar 10 μm . ( F ) Quantifications of the synapsin signal overlapping red beads . Data shown as means ± SEM ( n = 3 independent experiments , each data point corresponding to 58 - 171 analyzed beads ) , with comparisons made by two - way ANOVA with Holm - Sidak ' s posthoc test for multiple comparisons between the indicated groups .

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(E-F) Analysis of cerebellin(Cbln)-induced artificial synapse formation. (E) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured exposed to anti-V5-coupled beads pre-incubated with recombinant V5-tagged Cbln1 or empty anti-V5 beads. Cells were stained with antibodies against the synaptic marker synapsin (green), somatodendritic MAP2 (blue); beads are colored red and indicated by arrowheads. Scale bar 10 μm. (F) Quantifications of the synapsin signal overlapping red beads. Data shown as means ±SEM (n=3 independent experiments, each data point corresponding to 58-171 analyzed beads), with comparisons made by two-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups.


( A ) Representative image of a typical young WT cortico - hippocampal slice ( 350 µm ) from co - culture ( 14 DIV ) stained with DAPI ( gray ) to visualize nuclei ( left ) . Dentate gyrus ( DG ) , cornus ammonis 1 ( CA1 ) and cortical ( CTX ) regions are indicated . An example of the co - culture dish is shown on the right . Co - culture includes 2 young WT ( * , young co - culture ) and 2 old APPPS1 ( + , old co - culture ) brain slices .

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

(A) Representative image of a typical young WT cortico-hippocampal slice (350 µm) from co-culture (14 DIV) stained with DAPI (gray) to visualize nuclei (left). Dentate gyrus (DG), cornus ammonis 1 (CA1) and cortical (CTX) regions are indicated. An example of the co-culture dish is shown on the right. Co-culture includes 2 young WT (*, young co-culture) and 2 old APPPS1 (+, old co-culture) brain slices.


( B ) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone ( young alone , old alone ) or in co - culture ( young co - culture , old co - culture ) at 7 days in vitro ( DIV ) stained with propidium iodide ( PI , red ) reveals reduced cell viability in old brain slices compared to young . ( C ) Quantitative analysis of PI positive cells in young WT and old APPPS1 slices cultured alone ( white and black bars , respectively ) or in co - culture ( grey and white squared bars , respectively ) at 7 and 14 DIV . The values are expressed as percentages of PI positive cells from the total number of DAPI positive cells . The values represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( n . s . = not significant ; * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone (young alone, old alone) or in co-culture (young co-culture, old co-culture) at 7 days in vitro (DIV) stained with propidium iodide (PI, red) reveals reduced cell viability in old brain slices compared to young.(C) Quantitative analysis of PI positive cells in young WT and old APPPS1 slices cultured alone (white and black bars, respectively) or in co-culture (grey and white squared bars, respectively) at 7 and 14 DIV. The values are expressed as percentages of PI positive cells from the total number of DAPI positive cells. The values represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (n.s.= not significant; ***P < 0.001).


( D - F ) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone or in co - culture at 7 DIV and immunostained using neuronal ( NeuN ) , astrocytic ( GFAP ) and microglial ( CD68 ) markers ( green ) reveals reduced NeuN and GFAP and increased CD68 immunosignal . Scale bar : 75

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(D-F) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone or in co-culture at 7 DIV and immunostained using neuronal (NeuN), astrocytic (GFAP) and microglial (CD68) markers (green) reveals reduced NeuN and GFAP and increased CD68 immunosignal. Scale bar: 75 µm


( A ) Immunofluorescence analysis of the old APPPS1 slice in co - culture using antibody 6E10 to detect amyloid plaques ( green ) and Thiazine Red ( TR , red ) to visualize fibrillar amyloid cores . At 7 DIV , the majority of amyloid plaques contain 6E10 and TR positive dense core surrounded by 6E10 positive halo of diffuse Aβ . In contrast , at 14 DIV , the majority of amyloid plaques are core - only plaques co - stained with 6E10 and TR without detectable 6E10 positive halo . Lower panels are higher magnification images of boxed regions . Scale bars : 50 µm .

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

(A) Immunofluorescence analysis of the old APPPS1 slice in co-culture using antibody 6E10 to detect amyloid plaques (green) and Thiazine Red (TR, red) to visualize fibrillar amyloid cores. At 7 DIV, the majority of amyloid plaques contain 6E10 and TR positive dense core surrounded by 6E10 positive halo of diffuse Aβ. In contrast, at 14 DIV, the majority of amyloid plaques are core-only plaques co-stained with 6E10 and TR without detectable 6E10 positive halo. Lower panels are higher magnification images of boxed regions. Scale bars: 50 µm.


( B ) Quantitative analysis of core - only plaques in old APPPS1 slices cultured alone ( white bars ) or in co - culture ( gray bars ) at 7 and 14 DIV reveals an increased number of core - only plaques upon co - culturing of old APPPS1 brain slices together with young WT slices . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques . The values represent mean ± SEM from 3 independent experiments , each experiment including 3 independent slice culture dishes ( * * * P < 0 . 001 ) .

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

(B) Quantitative analysis of core-only plaques in old APPPS1 slices cultured alone (white bars) or in co-culture (gray bars) at 7 and 14 DIV reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young WT slices. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).


( C ) Western blot analysis of Aβ levels ( triplicates ) in the old APPPS1 tissue cultured alone or in co - culture with the young WT ( 14 DIV ) using 2D8 antibody . ( D ) Quantification of Western blot signals reveals decreased levels of Aβ upon co - culturing compared to old tissue cultured alone . The values are normalized to the levels of Aβ in the old tissue cultured alone and represent mean ± SEM from 2 independent experiments , each experiment was performed in triplicates ( * * P < 0 . 01 ) .

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(C) Western blot analysis of Aβ levels (triplicates) in the old APPPS1 tissue cultured alone or in co-culture with the young WT (14 DIV) using 2D8 antibody.(D) Quantification of Western blot signals reveals decreased levels of Aβ upon co-culturing compared to old tissue cultured alone. The values are normalized to the levels of Aβ in the old tissue cultured alone and represent mean ± SEM from 2 independent experiments, each experiment was performed in triplicates (**P < 0.01).


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

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


( F ) Immunofluorescence analysis of the old APPPS1 slice in co - culture ( 14 DIV ) treated with cytochalasin D ( CytoD ) and vehicle control ( Ctr ) and immunostained with M3 . 2 ( green ) and CD68 ( red ) . Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance . Scale bar : 50 µm . ( G ) Quantitative analysis of core - only plaques in the old APPPS1 slice in co - culture ( 14 DIV ) treated with CytoD and Ctr reveals a decreased number of core - only plaques upon CytoD treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques . The values represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * * P < 0 . 001 ) .

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(F) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) treated with cytochalasin D (CytoD) and vehicle control (Ctr) and immunostained with M3.2 (green) and CD68 (red). Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance. Scale bar: 50 µm.(G) Quantitative analysis of core-only plaques in the old APPPS1 slice in co-culture (14 DIV) treated with CytoD and Ctr reveals a decreased number of core-only plaques upon CytoD treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).


Either young WT ( A , B ) or old APPPS1 ( C - E ) tissue was treated with clodronate ( Clo ) to remove CD68 positive cells or vehicle Ctr from 1 until 7 DIV . Treatment was stopped and subsequently old ( A , B ) or young ( C - E ) tissue was added to the culture as schematically indicated in A and C . and analyzed 14 days after . Removal of CD68 positive cells in either young WT or old APPPS1 tissue prevents amyloid plaque clearance in the co - culture model . ( A ) Immunofluorescence analysis of the old APPPS1 slice co - cultured with the young WT slice pre - treated with Clo and Ctr and immunostained with CD68 ( red ) and M3 . 2 ( green ) . Scale bar : 50 µm . ( B ) Quantitative analysis of core - only plaques in the old APPPS1 tissue co - cultured with the young WT tissue pre - treated with Clo and Ctr as indicated in A reveals a decreased number of core - only plaques upon Clo treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * * P < 0 . 001 ) . ( C ) Immunofluorescence analysis of the old APPPS1 slice treated with Clo and Ctr and subsequently co - cultured with the young WT slice and immunostained with CD68 ( red ) and M3 . 2 ( green ) . Scale bar : 50 µm . ( D ) Area of CD68 positive cells ( CD68 coverage ) in the old APPPS1 tissue treated with Clo and Ctr and subsequently co - cultured with the young WT tissue as indicated in C . CD68 coverage is reduced upon Clo treatment . The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * * P < 0 . 001 ) . ( E ) Quantitative analysis of core - only plaques in the old APPPS1 tissue treated with Clo and Ctr and subsequently co - cultured with the young WT tissue as indicated in C reveals a decreased number of core - only plaques upon Clo treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * P < 0 . 01 ) .

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

Either young WT (A, B) or old APPPS1 (C-E) tissue was treated with clodronate (Clo) to remove CD68 positive cells or vehicle Ctr from 1 until 7 DIV. Treatment was stopped and subsequently old (A, B) or young (C-E) tissue was added to the culture as schematically indicated in A and C. and analyzed 14 days after. Removal of CD68 positive cells in either young WT or old APPPS1 tissue prevents amyloid plaque clearance in the co-culture model.(A) Immunofluorescence analysis of the old APPPS1 slice co-cultured with the young WT slice pre-treated with Clo and Ctr and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.(B) Quantitative analysis of core-only plaques in the old APPPS1 tissue co-cultured with the young WT tissue pre-treated with Clo and Ctr as indicated in A reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).(C) Immunofluorescence analysis of the old APPPS1 slice treated with Clo and Ctr and subsequently co-cultured with the young WT slice and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.(D) Area of CD68 positive cells (CD68 coverage) in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C. CD68 coverage is reduced upon Clo treatment. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).(E) Quantitative analysis of core-only plaques in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (**P < 0.01).


( A ) Immunofluorescence analysis of the old APPPS1 slice in co - culture with the young CX3CR1 + / GFP slice ( 14 DIV ) and immunostained using GFP ( green ) , CD68 ( red ) and M3 . 2 ( blue ) reveals no co - localization of young , GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques . A GFP antibody was used to amplify the signal of GFP - expressing young microglial cells . ( B ) Immunofluorescence analysis of the old APPPS1 / CX3CR1 + / GFP slice in co - culture with the young WT slice ( 14 DIV ) and immunostained using GFP ( green ) , CD68 ( red ) and M3 . 2 ( blue ) reveals co - localization of old , GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques . A GFP antibody was used to amplify the signal of GFP - expressing old microglial cells . Scale bar : 10 µm ( A and B ) .

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

(A) Immunofluorescence analysis of the old APPPS1 slice in co-culture with the young CX3CR1+/GFP slice (14 DIV) and immunostained using GFP (green), CD68 (red) and M3.2 (blue) reveals no co-localization of young, GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques. A GFP antibody was used to amplify the signal of GFP-expressing young microglial cells.(B) Immunofluorescence analysis of the old APPPS1/CX3CR1+/GFP slice in co-culture with the young WT slice (14 DIV) and immunostained using GFP (green), CD68 (red) and M3.2 (blue) reveals co-localization of old, GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques. A GFP antibody was used to amplify the signal of GFP-expressing old microglial cells. Scale bar: 10 µm (A and B).


( C ) 3D reconstruction of the old APPPS1 / CX3CR1 + / GFP slice in co - culture with the young WT slice ( 10 DIV ) and immunostained using GFP ( green ) and M3 . 2 ( red ) . GFP positive old microglial cells surrounding amyloid plaques exhibit intracellular M3 . 2 positive immunoreactivity indicative of Aβ uptake . Overview at low magnification is shown in the left panel . Scale bar : 15 µm . A detailed plaque is shown at higher magnification in the right panels . Scale bar : 10 µm .

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

(C) 3D reconstruction of the old APPPS1/CX3CR1+/GFP slice in co-culture with the young WT slice (10 DIV) and immunostained using GFP (green) and M3.2 (red). GFP positive old microglial cells surrounding amyloid plaques exhibit intracellular M3.2 positive immunoreactivity indicative of Aβ uptake. Overview at low magnification is shown in the left panel. Scale bar: 15 µm. A detailed plaque is shown at higher magnification in the right panels. Scale bar: 10 µm.


( D ) Immunofluorescence analysis of young CX3CR1 + / GFP and old APPPS1 / CX3CR1 + / GFP slices freshly cut ( 0 DIV ) , cultured alone or in co - culture ( 10 DIV ) and immunostained using GFP ( green ) shows alterations in microglial morphology of old cells upon culturing . Upper - left panels are enlargements of boxed regions . A GFP antibody was used to amplify the signal of GFP - expressing old microglial cells . Scale bars : 50 µm . ( E ) Quantitative analysis of amoeboid microglial cells in young CX3CR1 + / GFP ( white bars ) and old APPPS1 / CX3CR1 + / GFP ( green bars ) brain slices freshly cut ( 0 DIV ) , cultured alone or in co - culture ( 10 DIV ) . The values are expressed as percentages of amoeboid microglia from the total number of microglial cells . The values represent mean ± SEM from 3 independent experiments , each experiment including at least 250 cells per condition ( * P < 0 . 05 , * * * P < 0 . 001 ) .

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

(D) Immunofluorescence analysis of young CX3CR1+/GFP and old APPPS1/CX3CR1+/GFP slices freshly cut (0 DIV), cultured alone or in co-culture (10 DIV) and immunostained using GFP (green) shows alterations in microglial morphology of old cells upon culturing. Upper-left panels are enlargements of boxed regions. A GFP antibody was used to amplify the signal of GFP-expressing old microglial cells. Scale bars: 50 µm.(E) Quantitative analysis of amoeboid microglial cells in young CX3CR1+/GFP (white bars) and old APPPS1/CX3CR1+/GFP (green bars) brain slices freshly cut (0 DIV), cultured alone or in co-culture (10 DIV). The values are expressed as percentages of amoeboid microglia from the total number of microglial cells. The values represent mean ± SEM from 3 independent experiments, each experiment including at least 250 cells per condition (*P < 0.05, ***P < 0.001).


( A ) Old APPPS1 tissue was cultured alone ( old alone , white bars ) or incubated with conditioned media ( CM ) collected from young WT slices ( old alone + young CM , grey bars ) . Analysis included co - culture of old APPPS1 and young WT brain slices ( old co - culture , black bars ) as a positive control and old APPPS1 tissue incubated with conditioned media collected from old APPPS1 slices ( old alone + old CM , white squared bars ) as a negative control . Quantitative analysis at 7 , 11 and 14 DIV reveals increased numbers of core - only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices , fully recapitulating core - only plaque numbers observed in the co - culture . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including at least 2 independent slice culture dishes ( n . s . = not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Old APPPS1 tissue was cultured alone (old alone, white bars) or incubated with conditioned media (CM) collected from young WT slices (old alone + young CM, grey bars). Analysis included co-culture of old APPPS1 and young WT brain slices (old co-culture, black bars) as a positive control and old APPPS1 tissue incubated with conditioned media collected from old APPPS1 slices (old alone + old CM, white squared bars) as a negative control. Quantitative analysis at 7, 11 and 14 DIV reveals increased numbers of core-only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices, fully recapitulating core-only plaque numbers observed in the co-culture. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including at least 2 independent slice culture dishes (n.s.= not significant, **P <0.01, ***P < 0.001).


( B ) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices previously treated with clodronate ( old alone + young CM + Clo , grey bars ) and vehicle control ( old alone + young CM + Ctr , white bars ) . Quantitative analysis at 7 , 11 and 14 DIV reveals decreased numbers of core - only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices pre - treated with Clo . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including at least 2 independent slice culture dishes ( n . s . = not significant , * * * P < 0 . 001 ) .

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

(B) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices previously treated with clodronate (old alone + young CM + Clo, grey bars ) and vehicle control (old alone + young CM + Ctr, white bars). Quantitative analysis at 7, 11 and 14 DIV reveals decreased numbers of core-only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices pre-treated with Clo. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including at least 2 independent slice culture dishes (n.s.= not significant, ***P < 0.001).


( C ) Old APPPS1 tissue was incubated with conditioned media collected from cultured WT primary microglial cells ( old alone + MG CM , grey bar ) or with unconditioned slice culture media as a control ( old alone + Ctr , white bar ) . Quantitative analysis of core - only plaques at 14 DIV reveals enhanced amyloid plaque clearance upon incubation of the old APPPS1 tissue with conditioned media collected from cultured primary microglia . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * P < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Old APPPS1 tissue was incubated with conditioned media collected from cultured WT primary microglial cells (old alone + MG CM, grey bar) or with unconditioned slice culture media as a control (old alone + Ctr, white bar). Quantitative analysis of core-only plaques at 14 DIV reveals enhanced amyloid plaque clearance upon incubation of the old APPPS1 tissue with conditioned media collected from cultured primary microglia. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).


( A - F ) Immunofluorescence analysis of the old APPPS1 slice ( 14 DIV ) treated with vehicle Ctr in A , anti - inflammatory factors IL - 10 and TGF - β in B and C and pro - inflammatory factors IL - 6 , IL - 12 / p40 and GM - CSF in D , E and F , respectively and immunostained with CD68 ( red ) and M3 . 2 ( green ) . In contrast to increased number of core - only plaques upon GM - CSF treatment , other factors tested did not induce amyloid plaque clearance , resulting in a better preservation of the M3 . 2 positive halos of Aβ surrounding dense core of amyloid plaques . Scale bar : 50 µm .

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

(A-F) Immunofluorescence analysis of the old APPPS1 slice (14 DIV) treated with vehicle Ctr in A, anti-inflammatory factors IL-10 and TGF-β in B and C and pro-inflammatory factors IL-6, IL-12/p40 and GM-CSF in D, E and F, respectively and immunostained with CD68 (red) and M3.2 (green). In contrast to increased number of core-only plaques upon GM-CSF treatment, other factors tested did not induce amyloid plaque clearance, resulting in a better preservation of the M3.2 positive halos of Aβ surrounding dense core of amyloid plaques. Scale bar: 50 µm.


( A ) CD68 coverage in the old APPPS1 tissue ( 14 DIV ) treated with GM - CSF and vehicle Ctr . CD68 coverage is increased upon treatment with GM - CSF . The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * P < 0 . 05 ) .

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

(A) CD68 coverage in the old APPPS1 tissue (14 DIV) treated with GM-CSF and vehicle Ctr. CD68 coverage is increased upon treatment with GM-CSF. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).


( B ) Quantitative analysis of core - only plaques in the old APPPS1 tissue ( 14 DIV ) treated with GM - CSF and Ctr reveals an increased number of core - only plaques upon treatment with GM - CSF . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including 3 independent slice culture dishes ( * * * P < 0 . 001 ) .

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 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

(B) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) treated with GM-CSF and Ctr reveals an increased number of core-only plaques upon treatment with GM-CSF. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).


( C ) CD68 coverage in the old APPPS1 tissue ( 14 DIV ) incubated with conditioned media collected from young WT slices ( young CM ) or with non - conditioned slice culture media ( Ctr ) . CD68 coverage is increased upon incubation of the APPPS1 tissue with the young CM . The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * * P < 0 . 01 ) .

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

(C) CD68 coverage in the old APPPS1 tissue (14 DIV) incubated with conditioned media collected from young WT slices (young CM) or with non-conditioned slice culture media (Ctr). CD68 coverage is increased upon incubation of the APPPS1 tissue with the young CM. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (**P < 0.01).


( D ) Immunofluorescence analysis of the old APPPS1 slice cultured alone ( old alone ) or in co - culture with young GM - CSF - / - brain slices ( old co - culture ) at 14 DIV and immunostained with CD68 ( red ) and M3 . 2 ( green ) reveals an increased number of core - only plaques upon co - culturing with young GM - CSF - / - brain slices . Scale bar : 50 µm . ( E ) Quantitative analysis of core - only plaques in the old APPPS1 slice cultured alone or in co - culture with young GM - CSF - / - brain slices ( 14 DIV ) reveals an increased number of core - only plaques upon co - culturing of old APPPS1 brain slices together with young GM - CSF - / - brain slices . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques . The values represent mean ± SEM from 3 independent experiments , each experiment including 3 independent slice culture dishes ( * * * P < 0 . 001 ) . ( F ) CD68 coverage in the old APPPS1 tissue ( 14 DIV ) cultured alone or in co - culture with young GM - CSF - / - brain slices . CD68 coverage is increased upon co - culturing of old APPPS1 brain slices together with young GM - CSF - / - brain slices . The values are normalized to CD68 coverage of the old slice cultured alone and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * P < 0 . 05 ) .

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

(D) Immunofluorescence analysis of the old APPPS1 slice cultured alone (old alone) or in co-culture with young GM-CSF-/- brain slices (old co-culture) at 14 DIV and immunostained with CD68 (red) and M3.2 (green) reveals an increased number of core-only plaques upon co-culturing with young GM-CSF-/- brain slices. Scale bar: 50 µm.(E) Quantitative analysis of core-only plaques in the old APPPS1 slice cultured alone or in co-culture with young GM-CSF-/- brain slices (14 DIV) reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).(F) CD68 coverage in the old APPPS1 tissue (14 DIV) cultured alone or in co-culture with young GM-CSF-/- brain slices. CD68 coverage is increased upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are normalized to CD68 coverage of the old slice cultured alone and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).


( A ) Immunofluorescence analysis of the old APPPS1 slice ( 14 DIV ) treated with GM - CSF and vehicle Ctr and immunostained with CD68 ( red ) , cell proliferation marker Ki67 ( green ) and M3 . 2 ( blue ) . Scale bar : 50 µm . ( B ) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue ( 14 DIV ) treated with GM - CSF and Ctr reveals increased microglial proliferation upon treatment with GM - CSF . The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 2 independent experiments , each experiment including 4 independent slice culture dishes ( * * * P < 0 . 001 ) .

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

(A) Immunofluorescence analysis of the old APPPS1 slice (14 DIV) treated with GM-CSF and vehicle Ctr and immunostained with CD68 (red), cell proliferation marker Ki67 (green) and M3.2 (blue). Scale bar: 50 µm.(B) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue (14 DIV) treated with GM-CSF and Ctr reveals increased microglial proliferation upon treatment with GM-CSF. The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 2 independent experiments, each experiment including 4 independent slice culture dishes (***P < 0.001).


( C ) Immunofluorescence analysis of the old APPPS1 tissue cultured alone or in co - culture with young WT brain slices ( 14 DIV ) and immunostained with CD68 ( red ) , Ki67 ( green ) and M3 . 2 ( blue ) Scale bar : 50 µm . ( D ) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue cultured alone or in co - culture with young brain slices ( 14 DIV ) reveals increased microglial proliferation upon co - culturing of old and young brain slices . The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * * P < 0 . 01 ) .

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

(C) Immunofluorescence analysis of the old APPPS1 tissue cultured alone or in co-culture with young WT brain slices (14 DIV) and immunostained with CD68 (red), Ki67 (green) and M3.2 (blue) Scale bar: 50 µm.(D) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue cultured alone or in co-culture with young brain slices (14 DIV) reveals increased microglial proliferation upon co-culturing of old and young brain slices. The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (**P < 0.01).