E - G . Immunoblot of Rpl24 - GFP free GFP ( E ) , of total GFP relative to Pgk1 ( F ) , free GFP relative to Rpl24 - GFP ( G ) in stm1 ∆ , wild - type , STM1AAA , STM1EEE treated with rapamycin for 24 hours . At least six biological replicates are subjected to statistical using multiple unpaired t - . ( s . e . for short exposure ; l . e . for long exposure ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G. Immunoblot of Rpl24-GFP and free GFP (E), quantification of total GFP relative to Pgk1 (F), and free GFP relative to Rpl24-GFP (G) in stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests. (s.e. for short exposure; l.e. for long exposure).


J - K . Immunoblot ( J ) ( K ) of Rpl3 in wild - type stm1 ∆ without rapamycin , or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours . At least six biological replicates are subjected to statistical using multiple unpaired t - .

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 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

J-K. Immunoblot (J) and the quantification (K) of Rpl3 in wild-type and stm1∆ cells without rapamycin, or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.


A - B . Puromycin incorporation ( A ) its ( B ) in stm1 ∆ , wild - type , STM1AAA , STM1EEE starved of nitrogen for 24 hours restimulated with ( + N ) for 10 , 20 , or 30 minutes . Immunoblots for puromycin , Stm1 , Rpl3 , Pgk1 are shown . Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Puromycin incorporation (A) and its quantification (B) in stm1∆, wild-type, STM1AAA, and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids (+ N) for 10, 20, or 30 minutes. Immunoblots for puromycin, Stm1, Rpl3, and Pgk1 are shown. Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t-tests.


D - E . Representative microscopic for expression of Rpl24 - GFP ( D ) its ( E ) in tetrads of control ( RPL24GFP ) stm1 ∆ RPL24GFP ( n = 100 , each ) . Experiments are performed at least three times . Statistical is done by unpaired t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E. Representative fluorescent microscopic images for expression of Rpl24-GFP (D) and its intensity quantification (E) in tetrads of control (RPL24GFP) and stm1∆ RPL24GFP cells (n=100, each). Experiments are performed at least three times. Statistical analysis is done by unpaired t-test.


A - C . Polysome profile immunoblotting of SERBP1 , RPS6 , RPL7a across polysome fractions of HEK293T treated with DMSO ( A ) , INK128 ( B ) , or harringtonine ( C ) for 1 hour . Polysome profiles were performed using 5 - 50 % sucrose density gradients containing 150 mM KCl .

O 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-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

A-C. Polysome profile and immunoblotting of SERBP1, RPS6, and RPL7a across the polysome fractions of HEK293T cells treated with DMSO (A), INK128 (B), or harringtonine (C) for 1 hour. Polysome profiles were performed using 5-50% sucrose density gradients containing 150 mM KCl.


I - J . Immunoblot for ubiquitin ( I ) its ( J ) in supernatant ribosomal pellet obtained from HEK293T 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 - . Data are presented as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 hox - deletion strains in presence of 0 . 12 mM menadion ( MD ) , 7 mM H2O2 or 2 mM tert - butyl hydroperoxide ( tBooH ) . strains were incubated for 5 days at 28 ° C in .

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

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 photosensory domain in presence of HO

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

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 CitA - GFP ( ca . 170 from N - terminus of citrate synthase ) were expressed over night at 30 ° C in minimal medium with 2 % threonine 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 ) pellet 2 ( P2 ) with mitochondria were analyzed by Western Blot using anti GFP antibodies . CitA - GFPc = form ; CitA - GFPi = imported version .

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

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

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

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 HOXs along with photosensory domain of A . nidulans FphA ( PGP ) over night at 20 ° C . PGP was purified via Strep - tag system eluate concentrated ( upper panel ) . 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 ) eluate ( E ) . fractions were analyzed by Western Blot using Anti - His antibody ( lower panel ) .

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

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

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

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

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 , its derivatives harboring plasmids encoding MepM ,

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

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


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

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

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 digested by lysozyme ( upper panel ) or by lysozyme 5 µM MepM ( lower panel ) . Absorbance was monitored at 205 nm ( mAU , milli - absorbance unit ) .

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

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

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

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 188SLO - that was partially complemented from plasmid piSLO ( SLO - ( piSLO ) ) , 188 188SLO - that contained empty complementation vector , 188 ( pSIV4 ) SLO - ( pSIV4 ) , respectively .

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 JRS4 , JRS4SLO - ( SLO - ) , JRS4NADase - ( NADase - ) . Experiments were performed in triplicate values represent 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 association between EGFP - LC3 188 , 188SLO - ( SLO - ) , 188SLS - ( SLS - ) , or 188SLO - SLS - ( SLO - SLS - ) . Immunofluorescent distinguished intracellular ( Alexa - 568 , red ) from extracellular ( Alexa - 568 Alexa - 660 , red blue , respectively ) . Scale bar = 10 µm . of intracellular that were associated with EGFP - LC3 at 3 h post - infection is shown for each .

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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 strains 188 , 188SLO - , 188SLO - SLS - at 3 h post - infection . Arrowheads indicate presence of double or multiple membranes ( 188 188SLO - ) or single membranes ( 188SLO - SLS - ) partially or completely surrounding . Vacuolar compartments associated with 188 188SLO - also contain 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 strains 188 , 188SLO - , 188SLS - , 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 presence or absence of Beclin1 knockdown .

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


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

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.


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

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


A . Confocal microscopy demonstrating association between ubiquitin ( green ) intracellular ( red ) at 30 min post - infection in keratinocytes infected with 188 , 188SLO - , 188NADase - , or 188SLO ( Y255A ) . Extracellular were stained blue red for identification as described in Figure 2 . Scale bar = 10 µm . of intracellular that were associated with ubiquitin is indicated for each , on of at least 100 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 188 , 188SLO - , 188SLO ( Y255A ) . acid substitution Y255A in SLO abrogates ability of SLO to porate host , demonstrated by failure of culture supernatants from 188SLO ( Y255A ) to lyse erythrocytes despite producing wild - type amounts of variant SLO . 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 culture supernatants in cytosol of keratinocytes infected with 771 , 771SLO - , 771SLO ( Y255A ) , or 771NADase - . Intracellular NADase activity due to translocation by extracellular was identical between 771 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 . intracellular survival of 188SLO ( Y255A ) was significantly lower than that of its parental , 188 , 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 association between galectin 8 ( green ) intracellular ( red ) in keratinocytes infected for 30 min with 188 , 188SLO - , or 188 SLO - SLS - . Extracellular ( ( out ) ) were stained blue red for identification as described in Figure 2 . Scale bar = 10 µm . of intracellular that were associated with galectin 8 is indicated for each , on of at least 100 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 NADase inhibit lysosomal fusion to - containing autophagosomes in oropharyngeal keratinocytes . A . Confocal microscopy of keratinocytes infected with 188 , 188SLO - , or 188NADase - demonstrating association of lysosomal marker LAMP - 1 ( red ) with ( blue ) contained within EGFP - LC3 ( green ) - positive compartments at 1 h , 3 h , 6 h post - infection . Scale bar = 10 µm . B . of of within EGFP - LC3 - positive compartments that are co - localized with LAMP - 1 at 1 h , 3 h , 6 h . Data represent mean values from three independent experiments in which at least 100 intracellular 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 media subjected to heparinase treatment ( ' Heps ' ) . Samples were analyzed by reducing SDS / PAGE immunoblotting with antibodies against Fc - tag 3G10 epitope ( to detect HS stub which reveal HS after heparinase digestion ) . ( C ) of 3G10 ( reflecting HS ) signal normalized to that of Fc ( Nrxn1 ) . Bar graph shows means ± 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 : ( B ) Representative immunoblots ( of three independent experiments ) with antibodies against HA , 3G10 epitope ( for HS stub ) 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 : ( 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 ) cortical neurons : Cortical neurons were infected with control lentivirus ( empty vector ) or lentivirus expressing FLAG - tagged CA10 , lysates subjected to immunoprecipitation for neurexin heparinase treatment . ( F ) Samples were analyzed by immunoblotting using antibodies against neurexins , 3G10 ( for HS stub ) FLAG ( for CA10 ) . Stars indicate non - neurexin bands ( see results ) . ( G ) of low molecular neurexin ( LMW ; arrowhead ) in relation to total neurexin amounts [ upper band ( ) + LMW ] . Data shown as mean ± ( n = 3 independent experiments ) ; * , p < 0 . 05 by 2 - way ANOVA Holm - Sidak . ( H ) of 3G10 signal normalized to signal of corresponding neurexin band . Data shown as mean ± of biological replicates . * , p < 0 . 05 by 2 - way ANOVA Holm - Sidak ( 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 CA10 were immunoprecipitated from total mouse brain lysates subject to heparinase treatment . ( I ) Samples were analyzed by immunoblotting using antibodies against neurexins , 3G10 epitope ( for HS stub ) CA10 . Stars indicate non - neurexin bands ( see results ) . ( J ) of low molecular neurexin ( LMW ; arrowhead ) in relation to total neurexin amounts [ upper band ( ) + LMW ] . Data shown as mean ± ( n = 3 independent experiments ) ; * , p < 0 . 05 by 2 - way ANOVA Holm - Sidak . ( K ) of 3G10 signal normalized to signal of corresponding neurexin band . Data shown as mean ± of 3 biological replicates . * , p < 0 . 05 by 2 - way ANOVA Holm - Sidak .

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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 indicated harvested from HEK293 media subjected to heparinase treatment ( ' Heps ' ) . Samples were analyzed by reducing SDS / PAGE immunoblotting with antibodies against Fc ( for Nrxn1β ) , 3G10 epitope ( for HS stub ) V5 ( for CA10 ) . Input samples were analyzed separately for CA10 to ensure equal expression ( lower panel ) . ( C ) of 3G10 epitope signal , normalized to Fc ( Nrxn1β ) . Data shown are mean ± of 3 independent replicates . * , p < 0 . 05 ; by Mann - Whitney comparing each Nrxn1 mutant with 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 Fc - tag ( for Nrxn1β - Fc ) 3G10 epitope ( for HS stub ) V5 ( CA10 - V5 ) , as indicated . CA10 in input samples is shown for demonstration of equal expression . ( F , of 3G10 epitope signal normalized to Fc ( for Nrxn1β - Fc ) signals , Data shown are mean ± of 3 biological replicates . * , p < 0 . 05 ; * * * , p < 0 . 001 by 2 - way ANOVA Holm - Sidak .

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

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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 ) Nrxn1 stalk / CA10 ( black ) at a 2 : 1 molar ( 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 its glycosylated forms , from Nrxn1β expressed in HEK293 .

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


( D ) , expressed as % of total occupancy of each glycosylation form , upon control CA10 expression conditions . Bar graph shows means ± of 4 biological replicates ; * , p < 0 . 05 by Mann - Whitney comparing occupancy of each glycosylation with 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 cerebellum dissected from 8 - week - old mice . ( B ) of lower molecular neurexin ( LMW ; arrowhead ) in relation to total of α - neurexins [ upper band ( ) + LMW ] . Bar graphs show mean ± of 3 independent replicates * * * , p < 0 . 001 by 2 - way ANOVA Holm - Sidak , comparing to 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 ) cerebellum ( Cb ) using an anti - pan - neurexin antibody was heparinase - treated ( ' Heps ' ) analyzed by immunoblotting with antibodies against neurexin 3G10 monoclonal ( to detect HS stub ) . Stars indicate non - neurexin bands ( see results ) . ( D ) of low molecular neurexin ( LMW ; arrowhead ) in relation to total of α - neurexins [ upper band ( ) + LMW ] . Bar graphs show mean ± SEMof 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA Holm - Sidak comparing forebrain cerebellum . ( E ) of 3G10 signal normalized to signal of corresponding neurexin band . Bar graphs show mean ± of 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA Holm - Sidak comparing forebrain 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 ) 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 was quantified as Fc signal of normalized to a for endogenous neurexin ( n = 4 cultures ; three fields imaged averaged per well ) . Scatchard for LRRTM2 revealed an apparent Kd of 29 . 5 nM a Bmax of 0 . 49 nM for control Kd 35 . 3 nM , Bmax 0 . 28 nM upon expression of CA10 ; significant ( p = 0 . 0006 ) by non - linear regression . Data shown as mean ± 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 , analyzed as in ( A ) . apparent Kd for Nlgn1 was 186 nM Bmax 1 . 65 nM for control Kd 223 nM , Bmax 1 . 49 nM when CA10 was overexpressed . difference was not significant ( p = 0 . 20 ) by non - linear regression . Data shown as mean ± of biological replicates ( n = 4 ) .

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

(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 ) of cerebellin ( Cbln ) - induced artificial synapse formation . ( E ) Confocal of hippocampal neurons transduced to express CA10 - FLAG or control ( empty vector ) co - cultured exposed to anti - V5 - coupled beads pre - incubated with recombinant V5 - tagged Cbln1 or empty anti - V5 beads . were stained with antibodies against synaptic marker synapsin ( green ) , somatodendritic MAP2 ( blue ) ; beads are colored red indicated by arrowheads . Scale bar 10 μm . ( F ) Quantifications of synapsin signal overlapping red beads . Data shown as means ± ( 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 for multiple comparisons between indicated groups .

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

(E-F) 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 ) cortical ( CTX ) regions are indicated . An example of co - culture dish is shown on right . Co - culture includes 2 young WT ( * , young co - culture ) 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

(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 of young WT 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 viability in old brain slices compared to young . ( C ) Quantitative of PI positive in young WT old APPPS1 slices cultured alone ( black bars , respectively ) or in co - culture ( grey squared bars , respectively ) at 7 14 DIV . values are expressed as percentages of PI positive from total number of DAPI positive . values represent mean ± 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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 of old APPPS1 slice in co - culture using antibody 6E10 to detect amyloid plaques ( green ) Thiazine Red ( TR , red ) to visualize fibrillar amyloid cores . At 7 DIV , majority of amyloid plaques contain 6E10 TR positive dense surrounded by 6E10 positive halo of diffuse Aβ . In contrast , at 14 DIV , majority of amyloid plaques are - only plaques co - stained with 6E10 TR without detectable 6E10 positive halo . Lower panels are higher magnification 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 O O O O O O O O O O O O O O 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 of - only plaques in old APPPS1 slices cultured alone ( bars ) or in co - culture ( gray bars ) at 7 14 DIV reveals an increased number of - only plaques upon co - culturing of old APPPS1 brain slices together with young WT slices . values are expressed as percentages of - only plaques from total number of amyloid plaques . values represent mean ± 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

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

(C) Western blot analysis of 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 of old APPPS1 slice in co - culture ( 14 DIV ) using antibodies 6E10 ( green ) CD68 ( red ) . Nuclei were counterstained using DAPI ( blue ) . Image of boxed region in A is depicted at higher magnification in B reveals clustering of several CD68 positive around amyloid plaques . Scale bars : 100 µm ( A ) 10 µm ( B ) . ( C ) Quantitative of plaque - associated CD68 positive in co - culture samples at 7 14 DIV reveals a trend towards increased number of CD68 positive . values represent mean ± from at least 3 independent experiments , including total of at least 6 independent slice culture dishes ( n . s . = not significant ) . ( D ) Quantitative of co - culture samples at 7 14 DIV reveals a decrease in plaque . values represent mean ± from at least 3 independent experiments , including total of at least 6 replicates ( * * * P < 0 . 001 ) . ( E ) Quantitative of plaques surrounded by CD68 positive ( clustered plaques ) in co - culture samples at 7 14 DIV reveals an increase in number of clustered plaques . values are expressed as percentages of clustered plaques from total number of amyloid plaques . values represent mean ± from at least 3 independent experiments , including total of at least 6 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 of old APPPS1 slice in co - culture ( 14 DIV ) treated with cytochalasin D ( CytoD ) vehicle control ( Ctr ) immunostained with M3 . 2 ( green ) CD68 ( red ) . Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance . Scale bar : 50 µm . ( G ) Quantitative of - only plaques in old APPPS1 slice in co - culture ( 14 DIV ) treated with CytoD Ctr reveals a decreased number of - only plaques upon CytoD treatment . values are expressed as percentages of - only plaques from total number of amyloid plaques . values represent mean ± from 3 independent experiments , including total of 6 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 B-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 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 B-CONTROLLED_VAR 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

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

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

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

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

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

(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 , bars ) or incubated with conditioned media ( CM ) collected from young WT slices ( old alone + young CM , grey bars ) . included co - culture of old APPPS1 young WT brain slices ( old co - culture , black bars ) as a positive control old APPPS1 tissue incubated with conditioned media collected from old APPPS1 slices ( old alone + old CM , squared bars ) as a negative control . Quantitative at 7 , 11 14 DIV reveals increased numbers of - only plaques upon incubation of old APPPS1 tissue with conditioned media collected from young WT slices , fully recapitulating - only plaque numbers observed in co - culture . values are expressed as percentages of - only plaques from total number of amyloid plaques represent mean ± 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

(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 ) vehicle control ( old alone + young CM + Ctr , bars ) . Quantitative at 7 , 11 14 DIV reveals decreased numbers of - only plaques upon incubation of old APPPS1 tissue with conditioned media collected from young WT slices pre - treated with Clo . values are expressed as percentages of - only plaques from total number of amyloid plaques represent mean ± 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 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

(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 microglial ( old alone + MG CM , grey bar ) or with unconditioned slice culture media as a control ( old alone + Ctr , bar ) . Quantitative of - only plaques at 14 DIV reveals enhanced amyloid plaque clearance upon incubation of old APPPS1 tissue with conditioned media collected from cultured microglia . values are expressed as percentages of - only plaques from total number of amyloid plaques represent mean ± 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

(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 of old APPPS1 slice ( 14 DIV ) treated with vehicle Ctr in A , anti - inflammatory factors IL - 10 TGF - β in B C pro - inflammatory factors IL - 6 , IL - 12 / p40 GM - CSF in D , E F , respectively immunostained with CD68 ( red ) M3 . 2 ( green ) . In contrast to increased number of - only plaques upon GM - CSF treatment , other factors tested did not induce amyloid plaque clearance , resulting in a better preservation of M3 . 2 positive halos of Aβ surrounding dense 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 old APPPS1 tissue ( 14 DIV ) treated with GM - CSF vehicle Ctr . CD68 coverage is increased upon treatment with GM - CSF . values are normalized to CD68 coverage of Ctr represent mean ± 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

(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 of - only plaques in old APPPS1 tissue ( 14 DIV ) treated with GM - CSF Ctr reveals an increased number of - only plaques upon treatment with GM - CSF . values are expressed as percentages of - only plaques from total number of amyloid plaques represent mean ± 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

(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 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 APPPS1 tissue with young CM . values are normalized to CD68 coverage of Ctr represent mean ± 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of old APPPS1 slice cultured alone ( old alone ) or in co - culture with young GM - CSF - / - brain slices ( old co - culture ) at 14 DIV immunostained with CD68 ( red ) M3 . 2 ( green ) reveals an increased number of - only plaques upon co - culturing with young GM - CSF - / - brain slices . Scale bar : 50 µm . ( E ) Quantitative of - only plaques in old APPPS1 slice cultured alone or in co - culture with young GM - CSF - / - brain slices ( 14 DIV ) reveals an increased number of - only plaques upon co - culturing of old APPPS1 brain slices together with young GM - CSF - / - brain slices . values are expressed as percentages of - only plaques from total number of amyloid plaques . values represent mean ± from 3 independent experiments , each experiment including 3 independent slice culture dishes ( * * * P < 0 . 001 ) . ( F ) CD68 coverage in 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 . values are normalized to CD68 coverage of old slice cultured alone represent mean ± 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of old APPPS1 slice ( 14 DIV ) treated with GM - CSF vehicle Ctr immunostained with CD68 ( red ) , proliferation marker Ki67 ( green ) M3 . 2 ( blue ) . Scale bar : 50 µm . ( B ) Quantitative of Ki67 CD68 double positive microglial in old APPPS1 tissue ( 14 DIV ) treated with GM - CSF Ctr reveals increased microglial proliferation upon treatment with GM - CSF . values are expressed as percentages of CD68 Ki67 double positive microglial from total number of CD68 positive represent mean ± 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of old APPPS1 tissue cultured alone or in co - culture with young WT brain slices ( 14 DIV ) immunostained with CD68 ( red ) , Ki67 ( green ) M3 . 2 ( blue ) Scale bar : 50 µm . ( D ) Quantitative of Ki67 CD68 double positive microglial in old APPPS1 tissue cultured alone or in co - culture with young brain slices ( 14 DIV ) reveals increased microglial proliferation upon co - culturing of old young brain slices . values are expressed as percentages of CD68 Ki67 double positive microglial from total number of CD68 positive represent mean ± 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


( E ) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices in addition treated with proliferation inhibitor AraC ( young CM + AraC ) or vehicle Ctr ( young CM + Ctr ) . Immunofluorescence was performed at 14 DIV using CD68 ( red ) M3 . 2 ( green ) . Scale bar : 50 µm . ( F ) Quantitative of - only plaques in old APPPS1 tissue ( 14 DIV ) incubated with conditioned media from young WT slices in addition treated with AraC or Ctr reveals a decreased number of - only plaques upon AraC treatment . values are expressed as percentages of - only plaques from total number of amyloid plaques represent mean ± from 3 independent experiments , each including 2 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 B-CONTROLLED_VAR 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 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

(E) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices and in addition treated with proliferation inhibitor AraC (young CM + AraC) or vehicle Ctr (young CM + Ctr). Immunofluorescence analysis was performed at 14 DIV using CD68 (red) and M3.2 (green). Scale bar: 50 µm.(F) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) incubated with conditioned media from young WT slices and in addition treated with AraC or Ctr reveals a decreased number of core-only plaques upon AraC 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, each including 2 independent slice culture dishes (***P < 0.001).


A . Hcp release . HA - tagged Hcp ( HcpHA ) release was assessed by separating ( C ) - free culture supernatant ( S ) fractions from 109 wild - type ( WT ) , ΔtssM or ΔtssM carrying AHT - inducible FLAG - tagged tssM - borne plasmid ( tssM + ) treated ( bulgecin ) or not ( NT ) with bulgecin A prior to tssM gene induction . were separated by 12 . 5 % - acrylamide SDS - PAGE periplasmic TolB ( control for lysis ) , HcpHA FLTssM were immunodetected using anti - TolB ( middle panel ) , anti - HA ( lower panel ) anti - FLAG ( upper panel ) antibodies . Molecular markers ( in kDa ) are indicated on left . experiment was performed in duplicate a representative result is shown .

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

A. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT), ΔtssM cells or ΔtssM cells carrying the AHT-inducible FLAG-tagged tssM-borne plasmid (tssM+) treated (bulgecin) or not (NT) with bulgecin A prior to tssM gene induction. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis), HcpHA and FLTssM were immunodetected using anti-TolB (middle panel), anti-HA (lower panel) and anti-FLAG (upper panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.


B . Hcp release . HA - tagged Hcp ( HcpHA ) release was assessed by separating ( C ) - free culture supernatant ( S ) fractions from 109 wild - type ( WT ) before washing ( before wash ) after washing growth ( after wash ) in absence ( NT ) or presence ( bulgecin ) of bulgecin A . were separated by 12 . 5 % - acrylamide SDS - PAGE periplasmic TolB ( control for lysis ) HcpHA were immunodetected using anti - TolB ( upper panel ) anti - HA ( lower panel ) antibodies . Molecular markers ( in kDa ) are indicated on left . experiment was performed in duplicate a representative result is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT) cells before washing cells (before wash) and after washing and growth (after wash) in absence (NT) or presence (bulgecin) of bulgecin A. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis) and HcpHA were immunodetected using anti-TolB (upper panel) and anti-HA (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.


Hcp release . FLAG - tagged Hcp ( HcpFL ) release was assessed by separating ( C ) - free culture supernatant ( S ) fractions from 109 of indicated strains . were separated by 12 . 5 % - acrylamide SDS - PAGE TolB HcpFL were immunodetected using anti - TolB ( upper panel ) anti - FLAG ( lower panel ) antibodies . Molecular markers ( in kDa ) are indicated on left . experiment was performed in triplicate a representative result is shown .

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

Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 cells of the indicated strains. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB and HcpFL were immunodetected using anti-TolB (upper panel) and anti-FLAG (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.