( a ) microscopy of E . coli - containing phagosomes in macrophages transfected with RFP - conjugated LAMP - 1 , showing colocalization with E . coli - eGFP or S . aureus - eGFP . Right , of LAMP - 1 + phagosomes in microscopy at left .

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

(a) Fluorescence microscopy of E. coli-containing phagosomes in primary macrophages transfected with RFP-conjugated LAMP-1, showing colocalization with E. coli-eGFP or S. aureus-eGFP. Right, quantification of LAMP-1+ phagosomes in the microscopy at left.


( b ) Immunoblot of LAMP - 1 in phagosomes isolated by sucrose - gradient flotation from RAW264 . 7 macrophages after phagocytosis by Slamf1 - or mock - transfected RAW264 . 7 macrophages of beads coated with E . coli outer extract . WCL , whole - lysate . Right , of LAMP - 1 in immunoblot at left .

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

(b) Immunoblot analysis of LAMP-1 in phagosomes isolated by sucrose-gradient flotation from RAW264.7 macrophages after phagocytosis by Slamf1- or mock-transfected RAW264.7 macrophages of beads coated with E. coli outer membrane extract. WCL, whole-cell lysate. Right, quantification of LAMP-1 in the immunoblot at left.


( c ) Localization of MHC class II - eGFP ( MHCII GFP ) in phagosomes of macrophages from Slamf1 + / + or Slamf1 − / − MHC class II - eGFP ( B6 ) mice 3 - μm beads coated with E . coli outer extract . ( d ) Entry of 3 - μm beads coated with E . coli outer extract into lysosomes loaded with Texas red - dextran . Right ( c , d ) , of in microscopy at left . Numbers in bottom right corners ( a , c , d ) indicate time ( in min ) . Original magnification ( a - c ) , × 60 . DIC , differential interference contrast . Data are representative of three combined experiments ( a ) or at least three independent experiments ( b - d ) with at least 100 beads or 80 in each ( error bars , s . d . ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Localization of MHC class II-eGFP (MHCII GFP) in phagosomes of primary macrophages from Slamf1+/+ or Slamf1−/− MHC class II-eGFP (B6) mice and 3-μm beads coated with E. coli outer membrane extract. (d) Entry of 3-μm beads coated with E. coli outer membrane extract into lysosomes loaded with Texas red-dextran. Right (c,d), quantification of fluorescence in the microscopy at left. Numbers in bottom right corners (a,c,d) indicate time (in min). Original magnification (a-c), ×60. DIC, differential interference contrast. Data are representative of three combined experiments (a) or at least three independent experiments (b-d) with at least 100 beads or 80 bacteria in each (error bars, s.d.).


( a , b ) Association of EEA - 1 ( a ) or Rab5 ( b ) with phagosomes generated in macrophages by 3 - μm beads coated with E . coli outer extract ; numbers in bottom corners indicate time ( in min ) . α - , anti - . Original magnification , × 60 . Right ( a , b ) , of . Data are representative of at least three independent experiments ( error bars , s . d . ) .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Association of EEA-1 (a) or Rab5 (b) with phagosomes generated in primary macrophages by 3-μm beads coated with E. coli outer membrane extract; numbers in bottom corners indicate time (in min). α-, anti-. Original magnification, ×60. Right (a,b), quantification of fluorescence. Data are representative of at least three independent experiments (error bars, s.d.).


( a ) Colocalization of SLAM in RAW264 . 7 macrophages transfected with SLAM - mCherry allowed to phagocytose E . coli - eGFP or S . aureus - eGFP for 0 - 120 min ( time ( in min ) , bottom right corners at left ) . Original magnification , × 60 . Right , of at left . Data are representative of two independent experiments with a minimum of 40 per time point ( error bars , s . d . ) .

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

(a) Colocalization of SLAM and bacteria in RAW264.7 macrophages transfected with SLAM-mCherry and allowed to phagocytose E. coli-eGFP or S. aureus-eGFP for 0-120 min (time (in min), bottom right corners at left). Original magnification, ×60. Right, quantification of fluorescence at left. Data are representative of two independent experiments with a minimum of 40 bacteria per time point (error bars, s.d.).


( b ) Immunoblot of phagosome isolates from RAW264 . 7 macrophages transiently transfected with cDNA encoding Myc - tagged SLAM ( Slamf1 - Myc ) or mock transfected allowed to phagocytose beads coated with E . coli outer extract for 60 or 120 min . kDa , kilodaltons . Data are representative of at least three independent experiments .

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

(b) Immunoblot analysis of phagosome isolates from RAW264.7 macrophages transiently transfected with cDNA encoding Myc-tagged SLAM (Slamf1-Myc) or mock transfected and allowed to phagocytose beads coated with E. coli outer membrane extract for 60 or 120 min. kDa, kilodaltons. Data are representative of at least three independent experiments.


( a ) Luciferase activity in Jurkat transfected with a fusion of SLAM CD3ζ a luciferase reporter ( as described in Results ) , plus a renilla luciferase reporter , then exposed to heat - killed ( top ) .

O O O O O O O O O O O O O O O O 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) Luciferase activity in Jurkat cells transfected with a fusion of SLAM and CD3ζ and a luciferase reporter (as described in Results), plus a renilla luciferase reporter, then exposed to heat-killed bacteria (top).


( b ) Luciferase activity in Jurkat transfected with fusion in a ( Slamf1 - CD3ζ ) or a fusion of SLAM ectodomain construct lacking immunoglobulin V domain CD3ζ ( ΔIgV - Slamf1 - CD3ζ ) , luciferase reporters as in a , then exposed to heat - killed E . coli F18 .

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

(b) Luciferase activity in Jurkat cells transfected with the fusion in a (Slamf1-CD3ζ) or a fusion of SLAM ectodomain construct lacking the immunoglobulin V domain and CD3ζ (ΔIgV-Slamf1-CD3ζ), and luciferase reporters as in a, then exposed to heat-killed E. coli F18.


( c ) Luciferase activity in Jurkat transfected as in a , then left uninoculated ( − ) or inoculated with 10 × 108 E . coli F18 ( + ) , followed by addition of monoclonal antibody ( mAb ; amount , under graph ) 9D1 to SLAM ( α - SLAM ) or rat immunoglobulin G2b isotype - matched control antibody ( Rat IgG2b ) .

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

(c) Luciferase activity in Jurkat cells transfected as in a, then left uninoculated (−) or inoculated with 10 × 108 E. coli F18 (+), followed by the addition of monoclonal antibody (mAb; amount, under graph) 9D1 to SLAM (α-SLAM) or rat immunoglobulin G2b isotype-matched control antibody (Rat IgG2b).


( d ) Luciferase activity in Jurkat transfected as in a , then exposed to medium alone ( Med ) , wild - type E . coli ( JM101 ) , or JM101 E . coli mutants lacking both Omp C Omp F ( HN705 ) or lacking either OmpC ( ΔOmpC ) or OmpF ( ΔOmpF ) . Data are representative of at least three independent experiments ( error bars ( a ) , s . d . ) .

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

(d) Luciferase activity in Jurkat cells transfected as in a, then exposed to medium alone (Med), wild-type E. coli (JM101), or JM101 E. coli mutants lacking both Omp C and Omp F (HN705) or lacking either OmpC (ΔOmpC) or OmpF (ΔOmpF). Data are representative of at least three independent experiments (error bars (a), s.d.).


( a , b ) Production of phagosomal PtdIns ( 3 ) P in E . coli - containing phagosomes of peritoneal macrophages transfected with reporter cDNA encoding an eGFP - tagged PX domain of p40phox ( p40 PX - eGFP ) treated with DsRed - expressing E . coli ( E . coli - DsRed ; a ) or beads coated with E . coli outer extract ( b ) . Numbers in bottom right corners indicate time ( in min ) . Original magnification , × 60 . Right , of in microscopy at left .

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

(a,b) Production of phagosomal PtdIns(3)P in E. coli-containing phagosomes of primary peritoneal macrophages transfected with reporter cDNA encoding an eGFP-tagged PX domain of p40phox (p40 PX-eGFP) and treated with DsRed-expressing E. coli (E. coli-DsRed; a) or beads coated with E. coli outer membrane extract (b). Numbers in bottom right corners indicate time (in min). Original magnification, ×60. Right, quantification of fluorescence in microscopy at left.


( c ) HPLC of production of PtdIns ( 3 ) P in macrophages labeled with 3H - tagged myoinositol treated with beads coated with E . coli outer extract .

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

(c) HPLC analysis of the production of PtdIns(3)P in primary macrophages labeled with 3H-tagged myoinositol and treated with beads coated with E. coli outer membrane extract.


( d ) PtdIns ( 3 ) P production in RAW264 . 7 stably expressing SLAM or a mock construct , transfected with eGFP - tagged p40 PX treated with beads coated with E . coli outer extract . Data are from three combined experiments ( a ) or are representative of at least three independent experiments ( b , d ) or two independent experiments ( c ) with at least 100 beads or per experiment ( error bars , s . d . ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PtdIns(3)P production in RAW264.7 cells stably expressing SLAM or a mock construct, transfected with eGFP-tagged p40 PX and treated with beads coated with E. coli outer membrane extract. Data are from three combined experiments (a) or are representative of at least three independent experiments (b,d) or two independent experiments (c) with at least 100 beads or bacteria per experiment (error bars, s.d.).


( a , b ) Immunoassay of 293 transfected with various combinations of full - SLAM ( a ) or hemagglutinin - tagged ( - HA ) tail - less SLAM ( b ) , EAT - 2A , V5 - tagged ( - V5 ) Vps34 - Vps15 , beclin - 1 ; immunoprecipitated ( IP ) from lysates with monoclonal antibody to SLAM , as well as whole - lysates ( WCL ) , were analyzed by immunoblot ( IB ) with anti - beclin - 1 , anti - Vps34 , anti - V5 , anti - hemagglutinin , anti - SLAM or anti - EAT2 .

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

(a,b) Immunoassay of 293 cells transfected with various combinations of full-length SLAM (a) or hemagglutinin-tagged (-HA) tail-less SLAM (b), EAT-2A, V5-tagged (-V5) Vps34-Vps15, and beclin-1; proteins immunoprecipitated (IP) from lysates with monoclonal antibody to SLAM, as well as whole-cell lysates (WCL), were analyzed by immunoblot (IB) with anti-beclin-1, anti-Vps34, anti-V5, anti-hemagglutinin, anti-SLAM or anti-EAT2.


( c ) Microscopy of RAW264 . 7 transiently transfected with cDNA encoding GFP - tagged beclin - 1 SLAM - mCherry , then treated with beads coated with E . coli outer extract ( at a of 10 : 1 , beads / ) fixed after 60 min . Data are representative of six ( a ) or two ( b , c ) independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Microscopy of RAW264.7 cells transiently transfected with cDNA encoding GFP-tagged beclin-1 and SLAM-mCherry, then treated with beads coated with E. coli outer membrane extract (at a ratio of 10:1, beads/cells) and fixed after 60 min. Data are representative of six (a) or two (b,c) independent experiments.


Representative still of control , Rod depleted Rod depleted supplemented with RNAi resistant Venus - Rod treated with nocodazole time in minutes indicated . Scale bar , 5 μm .

O O O O O B-CONTROLLED_VAR 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

Representative still images of control, Rod depleted cells and Rod depleted cells supplemented with RNAi resistant Venus-Rod treated with nocodazole and time in minutes indicated. Scale bar, 5 μm.


Time from NEBD to mitotic exit for indicated conditions with each circle representing a single analyzed mean time indicated by red standard error of mean shown . number of counted mean time indicated above .

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

Time from NEBD to mitotic exit for the indicated conditions with each circle representing a single cell analyzed and mean time indicated by red line and standard error of mean shown. The number of cells counted and mean time indicated above.


Immunofluorescence of ZW10 Mad1 upon depletion of Rod . Scale bar , 5 μm .

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

Immunofluorescence analysis of ZW10 and Mad1 upon depletion of Rod. Scale bar, 5 μm.


of kinetochore intensities of indicated in control or Rod depleted with signal normalized to CREST Bub1 pSpT normalized to Bub1 . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments is shown .

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

Quantification of kinetochore intensities of the indicated proteins in control or Rod depleted cells with the signal normalized to CREST levels and Bub1 pSpT normalized to Bub1. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Time from NEBD to mitotic exit in control depleted , Rod depleted , Bub1 depleted Rod Bub1 co - depleted HeLa , U2OS RPE1 . were treated with nocodazole .

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

Time from NEBD to mitotic exit in control depleted, Rod depleted, Bub1 depleted and Rod and Bub1 co-depleted HeLa, U2OS and RPE1 cells. Cells were treated with nocodazole.


Mad1 kinetochore in indicated conditions normalized to level of CREST in HeLa . were fixed at 45 min after releasing from RO3306 arrest into nocodazole . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments is shown .

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

Mad1 kinetochore levels in the indicated conditions normalized to level of CREST in HeLa cells. Cells were fixed at 45 min after releasing from RO3306 arrest into nocodazole. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Western blot of whole extract for indicated conditions . Quantitative of Rod using LiCor technology is indicated below . asterisk indicates an unspecific band .

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

Western blot analysis of whole cell extract for the indicated conditions. Quantitative analysis of Rod levels using LiCor technology is indicated below. The asterisk indicates an unspecific band.


of ZW10 Mad1 kinetochore in indicated conditions . Kinetochore signal is normalized to CREST signal . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments shown .

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

Analysis of ZW10 and Mad1 kinetochore levels in the indicated cells and conditions. Kinetochore signal is normalized to CREST signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.


Representative still of unperturbed mitosis for parental , Rod C Rod CR with time in minutes indicated . CFP - Histone 3 was used here as chromosome marker . Scale bar , 5 μm .

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

Representative still images of unperturbed mitosis for parental cells, Rod C cells and Rod CR cells with time in minutes indicated. CFP-Histone 3 was used here as the chromosome marker. Scale bar, 5 μm.


Time from NEBD to exit of indicated conditions with treated with nocodazole Red circles represent still arrested in mitosis when filming stopped .

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

Time from NEBD to exit of indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit of indicated conditions with treated with taxol . Red circles represent still arrested in mitosis when filming stopped .

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

Time from NEBD to exit of indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit in indicated conditions with treated with nocodazole Red circles represent still arrested in mitosis when filming stopped .

Time from NEBD to exit in indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit in indicated conditions with treated with taxol . Red circles represent still arrested in mitosis when filming stopped .

Time from NEBD to exit in indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.


Mass spectrometry of BubR1 IPs from indicated lines . Relative values ( MaxLFQ , log10 ) are plotted across conditions . Data from of 3 technical repeats of BubR1 purifications with standard deviation indicated .

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

Mass spectrometry analysis of BubR1 IPs from the indicated cell lines. Relative protein quantification values (MaxLFQ, log10) are plotted across conditions. Data from analysis of 3 technical repeats of BubR1 purifications with standard deviation indicated.


Estimated of Bub1 in indicated conditions relative to parental lines . In Bub1 CR we only detected one peptide from three purifications . Standard deviation indicated .

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

Estimated protein levels of Bub1 in the indicated conditions relative to parental cell lines. In Bub1 CR we only detected one peptide from three purifications. Standard deviation indicated.


Representative still of unperturbed mitosis for parental , Bub1 C Bub1 CR with time in minutes indicated . CFP - Histone 3 was used as chromosome marker . Scale bar , 5 μm .

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

Representative still images of unperturbed mitosis for parental cells, Bub1 C cells and Bub1 CR cells with time in minutes indicated. CFP-Histone 3 was used as the chromosome marker. Scale bar, 5 μm.


Tethering of Mad1 ( Mad1 485 - 718 ) to Ndc80 time from NEBD to exit was measured in each condition . Red circles represent still arrested in mitosis when filming stopped .

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

Tethering of Mad1 (Mad1 485-718) to Ndc80 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped.


Tethering of Mad1 ( Mad1 485 - 718 ) to Ndc80 , KNL1 ( Zhang et al , 2017 ) or Bub1 1 - 553ΔCD1 time from NEBD to exit was measured in each condition . Red circles represent still arrested in mitosis when filming stopped .

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

Tethering of Mad1 (Mad1 485-718) to Ndc80, KNL1 (Zhang et al, 2017) or Bub1 1-553ΔCD1 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped.


Localization of Venus - tagged Mad1 in parental HeLa , Bub1 CR Rod CR in presence of nocodazole . Scale bar , 5 μm .

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

Localization of Venus-tagged Mad1 in parental HeLa cells, Bub1 CR cells and Rod CR cells in the presence of nocodazole. Scale bar, 5 μm.


Still from FRAP experiments using Venus - tagged Mad1 in indicated conditions . Image before bleach is shown then time following bleach . Arrowheads indicate kinetochore pairs . Scale bar , 5 μm for whole image 1 μm for zoom in on kinetochores .

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

Still images from FRAP experiments using Venus-tagged Mad1 in the indicated conditions. Image before bleach is shown and then time following bleach. Arrowheads indicate kinetochore pairs. Scale bar, 5 μm for whole cell image and 1 μm for the zoom in on kinetochores.


Analysis of Rod levels in cells used for FRAP experiment.


Venus - tagged Mad1 kinetochore versus time from bleaching shortly after NEBD in unperturbed ( left ) or in arrested with nocodazole ( right ) . Data combined from 3 independent experiments standard deviation indicated .

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

Venus-tagged Mad1 kinetochore intensity versus time from bleaching shortly after NEBD in unperturbed cells (left) or in cells arrested with nocodazole (right). Data combined from 3 independent experiments and standard deviation indicated.


A HeLa stably expressing Mad1 - BirA was depleted of Rod synchronized in mitosis using thymidine nocodazole . Biotin was added to media where indicated biotinylated were purified analyzed by western blot with indicated antibodies . Representative of 2 independent experiments .

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

A HeLa cell line stably expressing Mad1-BirA was depleted of Rod and synchronized in mitosis using thymidine and nocodazole. Biotin was added to media where indicated and biotinylated proteins were purified and analyzed by western blot with the indicated antibodies. Representative of 2 independent experiments.


duration from NEBD to exit in each indicated conditions was recorded by time - lapse microscopy .

The duration from NEBD to exit in each indicated conditions was recorded by time-lapse microscopy.


Mitotic duration in indicated conditions expressing Mad1 - Bub1 ( Mad1 485 - 718 fused to Bub1 1 - 553ΔCD1 ) fusion . time from NEBD to exit is indicated . Red circles represent still arrested in mitosis when filming stopped .

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

Mitotic duration in the indicated conditions expressing Mad1-Bub1 (Mad1 485-718 fused to Bub1 1-553ΔCD1) fusion protein. The time from NEBD to exit is indicated. Red circles represent cells still arrested in mitosis when the filming stopped.


depleted of Rod using RNAi transfected with indicated Bub1 constructs . ( D ) Representative immunofluorescence of stained for YFP Mad1 in each condition . Scale bar , 5 μm .

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

Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. (D) Representative immunofluorescence images of cells stained for YFP and Mad1 in each condition. Scale bar, 5 μm.


depleted of Rod using RNAi transfected with indicated Bub1 constructs . Mad1 kinetochore were measured normalized to Bub1 ( YFP ) level . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments is shown .

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

Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. Mad1 kinetochore levels were measured and normalized to Bub1 (YFP) level. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Bub1 CR complemented with indicated Bub1 constructs time from NEBD to exit in nocodazole measured by time - lapse microscopy . Mean ( red ) standard error of mean ( black bar ) indicated .

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

Bub1 CR cells complemented with the indicated Bub1 constructs and time from NEBD to exit in nocodazole measured by time-lapse microscopy. Mean (red line) and standard error of mean (black bar) indicated.


Kinetochore of Mad1 in Bub1 CR complemented with indicated YFP tagged Bub1 constructs . Mad1 are normalized to YFP signal . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments shown .

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

Kinetochore levels of Mad1 in Bub1 CR cells complemented with the indicated YFP tagged Bub1 constructs. Mad1 levels are normalized to YFP signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.


Still of unperturbed mitosis of HeLa transfected with Venus - Bub1 4xCD1 . CFP - Histone 3 was used as chromosome marker ( middle panel ) Venus - Bub1 4xCD1 was shown in bottom panel . Scale bar , 5 μm .

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

Still images of unperturbed mitosis of HeLa cells transfected with Venus-Bub1 4xCD1. CFP-Histone 3 was used as the chromosome marker (the middle panel) and Venus-Bub1 4xCD1 was shown in the bottom panel. Scale bar, 5 μm.


HeLa transfected with indicated Bub1 constructs time from NEBD to exit determined during an unperturbed mitosis . time from NEBD to exit was analyzed in each condition . Mean ( red ) standard error of mean ( black bar ) indicated . ( Mann Whitney , ns : non - significant ) . A representative result from at least 3 independent experiments is shown .

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

HeLa cells transfected with the indicated Bub1 constructs and time from NEBD to exit determined during an unperturbed mitosis. The time from NEBD to exit was analyzed in each condition. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ns: non-significant). A representative result from at least 3 independent experiments is shown.


Localization of Venus - Rod during unperturbed mitosis in control depleted or Bub1 depleted .

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

Localization of Venus-Rod during unperturbed mitosis in control depleted or Bub1 depleted cells.


HeLa were depleted of Bub1 using RNAi complemented with indicated Venus - Bub1 constructs . Immunofluorescence were shown for Bub1 ZW10 localization . of Bub1 ZW10 kinetochore normalized to CREST in indicated conditions .

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

HeLa cells were depleted of Bub1 using RNAi and complemented with indicated Venus-Bub1 constructs. Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions.


HeLa were depleted of Bub1 using RNAi Immunofluorescence were shown for Bub1 ZW10 localization . of Bub1 ZW10 kinetochore normalized to CREST in indicated conditions . with indicated Bub1 fusion

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

HeLa cells were depleted of Bub1 using RNAi Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions. with the indicated Bub1 fusion proteins


A Neurosphere assays were performed using mouse E14 . 5 neural progenitors treated with CRH with or without antalarmin ( Ant ) ( n = 3 biological replicates ) .

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

A Neurosphere assays were performed using mouse E14.5 primary neural progenitors treated with CRH with or without antalarmin (Ant) (n = 3 biological replicates).


B Effects of CRH treatment on stemness of neural progenitor at 2 days post - treatment were analyzed by Western blotting using an anti - SOX2 antibody ( n = 3 biological replicates ) .

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

B Effects of CRH treatment on the stemness of neural progenitor cells at 2 days post-treatment were analyzed by Western blotting using an anti-SOX2 antibody (n = 3 biological replicates).


Effects of CRHR1 overexpression on ( C ) neurosphere

Effects of CRHR1 overexpression on the (C) neurosphere formation


Effects of CRHR1 overexpression on ( D ) SOX2 expression ( n = 3 biological replicates ) .

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

Effects of CRHR1 overexpression on the (D) SOX2 expression (n = 3 biological replicates).


E CRHR1 knockdown efficiency of shRNAs used in this study ( n = 4 biological replicates ) .

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

E CRHR1 knockdown efficiency of shRNAs used in this study (n = 4 biological replicates).


Effects of CRHR1 knockdown on ( F ) neurosphere formation ,

Effects of CRHR1 knockdown on the (F) neurosphere formation,


Effects of CRHR1 knockdown on ( G ) SOX2 expression n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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

Effects of CRHR1 knockdown on the (G) SOX2 expression n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


Effects of CRHR1 knockdown on ( H ) neurosphere , n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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

Effects of CRHR1 knockdown on the (H) neurosphere morphology, n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


Effects of CRHR1 knockdown on ( I ) βIII - tubulin expression . n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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

Effects of CRHR1 knockdown on the (I) βIII-tubulin expression. n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


J , K Immunostaining using anti - GFP ( green ) together with ( J ) anti - βIII - tubulin ( clone TuJ1 ; red ) ( n = 5 biological replicates ) or ( K ) anti - GFAP ( red ) antibodies ( n = 3 biological replicates ) after in vitro differentiation of E14 . 5 neural progenitor transduced with a lentiviral vector expressing shCRHR1 .

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

J, K Immunostaining using anti-GFP (green) together with (J) anti-βIII-tubulin (clone TuJ1; red) (n = 5 biological replicates) or (K) anti-GFAP (red) antibodies (n = 3 biological replicates) after in vitro differentiation of E14.5 neural progenitor cells transduced with a lentiviral vector expressing shCRHR1.


A Embryonic brains that were intraventricularly electroporated CRHR1 - expressing plasmids at E13 . 5 were harvested at E15 . 5 immunostained using an anti - GFP ( green ) SOX2 ( red ) antibodies . B of fraction in each laminar layer in ( A ) ( n = 3 biological replicates ) . C of SOX2 + among GFP + in ( A ) ( n = 4 biological replicates ) .

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

A Embryonic brains that were intraventricularly electroporated CRHR1-expressing plasmids at E13.5 were harvested at E15.5 and immunostained using an anti-GFP (green) and SOX2 (red) antibodies. B Quantification of the cell fraction in each laminar layer in (A) (n = 3 biological replicates). C Percentage of SOX2+ cells among GFP+ cells in (A) (n = 4 biological replicates).


D Embryonic brains were electroporated with shCRHR1 plasmids with or without shCRHR1 - resistant CRHR1 expression vectors at E14 . 5 , samples were harvested at E17 . 5 for anti - GFP immunolabeling . GFP + localization was quantified ( bottom panel ) ( n = 5 biological replicates ) .

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

D Embryonic brains were electroporated with shCRHR1 plasmids with or without shCRHR1-resistant CRHR1 expression vectors at E14.5, and samples were harvested at E17.5 for anti-GFP immunolabeling. GFP+ cell localization was quantified (bottom panel) (n = 5 biological replicates).


E , F Brains electroporated with CRHR1 expression vectors at E13 . 5 were harvested at E15 . 5 co - immunostained using anti - GFP ( green ) together with ( E ) anti - TBR2 ( red ) or ( F ) anti - CTIP2 ( red ) antibodies . n = 3 biological replicates .

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

E, F Brains electroporated with CRHR1 expression vectors at E13.5 were harvested at E15.5 and co-immunostained using anti-GFP (green) together with (E) anti-TBR2 (red) or (F) anti-CTIP2 (red) antibodies. n = 3 biological replicates.


G E14 . 5 Embryonic brains that were electroporated with CRHR1 - expression vectors at E13 . 5 were labeled with EdU harvested at E15 . 5 . Samples were then visualized for GFP ( green ) , EdU ( blue ) Ki67 ( red ) ( n = 4 biological replicates ) .

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

G E14.5 Embryonic brains that were electroporated with CRHR1-expression vectors at E13.5 were pulse labeled with EdU and harvested at E15.5. Samples were then visualized for GFP (green), EdU (blue) and Ki67 (red) (n = 4 biological replicates).


of effects of CRHR1 knockdown on neural stemness - - fate - regulating transcription factors in neural progenitor by ( A ) qPCR at 2 days post - transduction .

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

Analysis of the effects of CRHR1 knockdown on neural stemness- and cell-fate-regulating transcription factors in neural progenitor cells by (A) qPCR at 2 days post-transduction.


of effects of CRHR1 knockdown on neural stemness - - fate - regulating transcription factors in neural progenitor by ( B ) Western blotting ( n = 3 biological replicates ) at 2 days post - transduction .

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

Analysis of the effects of CRHR1 knockdown on neural stemness- and cell-fate-regulating transcription factors in neural progenitor cells by (B) Western blotting (n = 3 biological replicates) at 2 days post-transduction.


effects of CRHR1 overexpression on expression in neural progenitor analyzed by

The effects of CRHR1 overexpression on REST expression in neural progenitor cells analyzed by qPCR


effects of CRHR1 overexpression on expression in neural progenitor analyzed by ( B ) Western blotting ( n = 6 biological replicates ) .

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

The effects of CRHR1 overexpression on REST expression in neural progenitor cells analyzed by (B) Western blotting (n = 6 biological replicates).


E Double - immunolabeling of E15 . 5 brain sections electroporated in utero with CRHR1 - expressing plasmid with or without shREST vectors at E13 . 5 using anti - GFP ( green ) SOX2 ( red ) antibodies .

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-MEASURED_VAR O O O O O

E Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid with or without shREST vectors at E13.5 using anti-GFP (green) and SOX2 (red) antibodies.


F , G of ( F ) GFP + distribution ( n ≥ 4 biological replicates ) ( G ) of SOX2 + ( n ≥ 3 biological replicates ) in ( E ) .

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

F, G Quantification of (F) GFP+ cell distribution (n ≥ 4 biological replicates) and (G) percentage of SOX2+ cells (n ≥ 3 biological replicates) in (E).


H Embryonic brains were electroporated with shCRHR1 plasmids with or without expression vectors at E14 . 5 , brain samples were harvested at E17 . 5 for anti - GFP immunolabeling . I of GFP + localization in ( 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

H Embryonic brains were electroporated with shCRHR1 plasmids with or without REST expression vectors at E14.5, and brain samples were harvested at E17.5 for anti-GFP immunolabeling. I Quantification of GFP+ cell localization in (H)


A , B Western blot of E14 . 5 neural progenitor transduced with a lentiviral vector expressing ( A ) CRHR1 or ( B ) shCRHR1 using an anti - CREB anti - phospho - CREB ( pCREB ) ( S133 ) antibodies . n = 5 biological replicates .

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

A, B Western blot analysis of E14.5 neural progenitor cells transduced with a lentiviral vector expressing (A) CRHR1 or (B) shCRHR1 using an anti-CREB and anti-phospho-CREB (pCREB) (S133) antibodies. n = 5 biological replicates.


C E14 . 5 neural progenitor were transduced with CRHR1 - containing lentiviral vectors . At 2 days post - transduction , were treated with 10 µM H89 ( PKA inhibitor ) or 1 µM KN93 ( CaM kinase inhibitor ) for 8 h harvested for Western blot for CREB ( top panel ) . Band intensities were quantified ( bottom panel ) ( n = 3 biological replicates ) .

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

C E14.5 neural progenitor cells were transduced with CRHR1-containing lentiviral vectors. At 2 days post-transduction, cells were treated with 10 µM H89 (PKA inhibitor) or 1 µM KN93 (CaM kinase inhibitor) for 8 h and harvested for Western blot analysis for CREB (top panel). Band intensities were quantified (bottom panel) (n = 3 biological replicates).


D Double - immunolabeling of E15 . 5 brain sections electroporated in utero with CRHR1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) pCREB ( S133 ) ( red ) as antibodies . Immunostaining was quantified ( right panel ) ( n = 5 biological replicates ) . inset boxes were magnified shown in panels D ' D ' ' .

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

D Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid at E13.5 using anti-GFP (green) and pCREB (S133) (red) as primary antibodies. Immunostaining was quantified (right panel) (n = 5 biological replicates). White inset boxes were magnified and shown in the panels D' and D''.


E Double - immunolabeling of E15 . 5 brain sections electroporated in utero with CRHR1 - expressing plasmid with or without a vector expressing CREB S133A , a dominant negative mutant form of CREB , at E13 . 5 using anti - GFP ( green ) SOX2 ( red ) antibodies .

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 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 B-MEASURED_VAR O O O O O

E Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid with or without a vector expressing CREB S133A, a dominant negative mutant form of CREB, at E13.5 using anti-GFP (green) and SOX2 (red) antibodies.


F , G of ( F ) GFP + distribution ( n = 4 biological replicates ) ( G ) of SOX2 + ( n ≥ 3 biological replicates ) in ( E ) .

F, G Quantification of (F) GFP+ cell distribution (n = 4 biological replicates) and (G) percentage of SOX2+ cells (n ≥ 3 biological replicates) in (E).


H Embryonic brains were electroporated with shCRHR1 plasmids with or without CREB expression vectors at E14 . 5 , then brain samples were harvested at E17 . 5 for anti - GFP immunolabeling . I of GFP + localization in ( L ) ( n ≥ 4 biological replicates ) .

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

H Embryonic brains were electroporated with shCRHR1 plasmids with or without CREB expression vectors at E14.5, and then brain samples were harvested at E17.5 for anti-GFP immunolabeling. I Quantification of GFP+ cell localization in (L) (n ≥ 4 biological replicates).


( A ) qPCR of E14 . 5 neural progenitor transduced with CREB - expressing lentiviral vectors , measuring

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

(A) qPCR of E14.5 neural progenitor cells transduced with CREB-expressing lentiviral vectors, measuring REST mRNA


( B ) Western blot analyses ( n = 3 biological replicates ) of E14 . 5 neural progenitor transduced with CREB - expressing lentiviral vectors , measuring , respectively .

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

(B) Western blot analyses (n = 3 biological replicates) of E14.5 neural progenitor cells transduced with CREB-expressing lentiviral vectors, measuring REST protein levels, respectively.


Dose - dependent inhibition of endogenous expression by a CREB inhibitor , 666 - 15 . E14 . 5 neural progenitor were treated with indicated concentrations of 666 - 15 for 48 h subjected to ( C )

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

Dose-dependent inhibition of endogenous REST expression by a CREB inhibitor, 666-15. E14.5 neural progenitor cells were treated with the indicated concentrations of 666-15 for 48 h and subjected to (C) qPCR


Dose - dependent inhibition of endogenous expression by a CREB inhibitor , 666 - 15 . E14 . 5 neural progenitor were treated with indicated concentrations of 666 - 15 for 48 h subjected to ( D ) Western blot

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

Dose-dependent inhibition of endogenous REST expression by a CREB inhibitor, 666-15. E14.5 neural progenitor cells were treated with the indicated concentrations of 666-15 for 48 h and subjected to (D) Western blot analyses


Inhibition of CRHR1 - induced expression by 666 - 15 . E14 . 5 neural progenitor transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666 - 15 , after 48 h , were lysed subjected to ( E )

O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

Inhibition of CRHR1-induced REST expression by 666-15. E14.5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666-15, and after 48 h, cells were lysed and subjected to (E) qPCR


Inhibition of CRHR1 - induced expression by 666 - 15 . E14 . 5 neural progenitor transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666 - 15 , after 48 h , were lysed subjected to ( F ) Western blot analyses ( n = 4 biological replicates ) .

O O B-CONTROLLED_VAR 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 B-MEASURED_VAR 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

Inhibition of CRHR1-induced REST expression by 666-15. E14.5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666-15, and after 48 h, cells were lysed and subjected to (F) Western blot analyses (n = 4 biological replicates).


H Luciferase activity was measured using E14 . 5 neural progenitor transfected with CREB expression vectors pGL3 - Basic luciferase plasmids harboring wild - type or mutated promoter at 2 days post - transfection ( n = 3 biological replicates ) .

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

H Luciferase activity was measured using E14.5 neural progenitor cells transfected with CREB expression vectors and pGL3-Basic luciferase plasmids harboring the wild-type or mutated REST promoter at 2 days post-transfection (n = 3 biological replicates).


I A representative gel image of ChIP showing association of CREB with regions of promoter by PCR amplification of putative CRE sequences in promoter ( n = 3 biological replicates ) .

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

I A representative gel image of ChIP analysis showing association of CREB proteins with regions of the REST promoter by PCR amplification of putative CRE sequences in the REST promoter (n = 3 biological replicates).


J Immunolabeling of E15 . 5 brain sections electroporated in utero with CREB - expressing plasmids with or without shREST vectors at E13 . 5 using anti - GFP antibody . GFP + localization was quantified ( right panel ) ( n ≥ 5 biological replicates ) . upper lower dashed lines indicate boundaries between CP IZ , IZ VZ / SVZ , respectively .

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

J Immunolabeling of E15.5 brain sections electroporated in utero with CREB-expressing plasmids with or without shREST vectors at E13.5 using anti-GFP antibody. GFP+ cell localization was quantified (right panel) (n ≥ 5 biological replicates). The upper and lower dashed lines indicate the boundaries between the CP and IZ, and IZ and VZ/SVZ, respectively.


3 ‐ month ‐ old wild ‐ type ( C57BL / 6 ) , Ltbr − / − , Lta − / − , − / − , Ltbr − / − Hox11 − / − mice ; of mice with at least one ANA .

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

3‐month‐old wild‐type (C57BL/6), Ltbr−/−, Lta−/−, LIGHT−/−, and Ltbr−/−Hox11−/− mice; percentage of mice with at least one ANA.


6 ‐ month ‐ old Ltbr − / − mice ( n = 15 ) ; titers of mice .

6‐month‐old Ltbr−/− mice (n = 15); titers of individual mice.


LIA of 6 ‐ month ‐ old Ltbr − / − mice , wild ‐ type mice , 3 ‐ month ‐ old Rorγt ‐ Ltb − / − mice .

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

LIA of 6‐month‐old Ltbr−/− mice, wild‐type mice, and 3‐month‐old Rorγt‐Ltb−/− mice.


LTβR ‐ Fc or control immunoglobulin ( Ig ) ‐ treated wild ‐ type mice ; of mice with at least one ANA . E denotes gestational day . * ANA were determined with LIA at of 3 months . * * ANA were determined with LIA 3 months after start of treatment .

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

LTβR‐Fc or control immunoglobulin (Ig)‐treated wild‐type mice; percentage of mice with at least one ANA. E denotes gestational day. *ANA were determined with LIA at the age of 3 months. **ANA were determined with LIA 3 months after the start of treatment.


3 ‐ month ‐ old ‐ specific LT knockout mice ; of mice with at least one ANA .

3‐month‐old cell‐specific LT knockout mice; percentage of mice with at least one ANA.


Bone marrow from wild ‐ type or Ltbr − / − mice were transferred at neonatal into lethally irradiated wild ‐ type or Ltbr − / − mice ; of mice with at least one ANA . ANA were determined with LIA at of 3 months .

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

Bone marrow cells from wild‐type or Ltbr−/− mice were transferred at neonatal age into lethally irradiated wild‐type or Ltbr−/− mice; percentage of mice with at least one ANA. ANA were determined with LIA at the age of 3 months.


Thymic reconstitution was analyzed by counting total number flow cytometry for percentages . Numbers represent percentages within indicated regions ( left panel ) . No significant differences were found . Data represent means ± s . e . m .

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

Thymic reconstitution was analyzed by counting total cell number and flow cytometry for cell percentages. Numbers represent the percentages within the indicated regions (left panel). No significant differences were found. Data represent means ± s.e.m.


T ‐ reconstitution of spleen was assessed by flow cytometry . Numbers represent percentages within indicated regions .

T‐cell reconstitution of spleen was assessed by flow cytometry. Numbers represent the percentages within the indicated regions.


Total IgG from nude mice engrafted with Ltbr − / − or wild ‐ type thymi was determined by ELISA . No significant differences were found . Data represent means ± s . e . m .

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

Total IgG from nude mice engrafted with Ltbr−/− or wild‐type thymi was determined by ELISA. No significant differences were found. Data represent means ± s.e.m.


Sera were collected 3 months after transplantation tested for ANA ; of mice with at least one ANA reactivity .

Sera were collected 3 months after transplantation and tested for ANA; percentage of mice with at least one ANA reactivity.


Ltbr − / − mice were treated or not with from birth ( upper panel ) , conventionalized or germfree wild ‐ type mice were treated with LTβR ‐ Fc fusion from gestational day 18 until 6 after birth ( lower panel ) ; of mice with at least one ANA . ANA were tested with LIA at of 3 months .

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

Ltbr−/− mice were treated or not with antibiotics from birth (upper panel), conventionalized or germfree wild‐type mice were treated with the LTβR‐Fc fusion protein from gestational day 18 until 6 weeks after birth (lower panel); percentage of mice with at least one ANA. ANA were tested with LIA at the age of 3 months.


Real ‐ time PCR for SFB in luminal , mucosal , fecal samples of multiple ANA ‐ positive ANA ‐ negative Ltbr − / − mice . with ANOVA , P = 0 . 02 for differences between groups , n = 4 mice per group . Data represent means ± s . e . m .

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

Real‐time PCR for SFB in luminal, mucosal, and fecal samples of multiple ANA‐positive and ANA‐negative Ltbr−/− mice. Analysis with ANOVA, P = 0.02 for differences between groups, n = 4 mice per group. Data represent means ± s.e.m.


C3H / HeN germfree pregnant mice were treated with LTβR ‐ Fc fusion on day 16 18 of gestation to prevent development of PP in their offspring . Pups were also treated in neonatal period to prevent development of ILF . Mice were colonized at 8 of with SFB or E . coli were sacrificed on d20 or on d60 postcolonization ; of mice with at least one ANA . E denotes gestational day .

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

C3H/HeN germfree pregnant mice were treated with the LTβR‐Fc fusion protein on day 16 and 18 of gestation to prevent the development of PP in their offspring. Pups were also treated in the neonatal period to prevent the development of ILF. Mice were colonized at 8 weeks of age with SFB or E. coli and were sacrificed on d20 or on d60 postcolonization; percentage of mice with at least one ANA. E denotes gestational day.


LTβR ‐ Fc ‐ treated wild ‐ type or IL17R − / − mice ; of mice with at least one ANA . E denotes gestational day . ANA were determined with LIA at of 3

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

LTβR‐Fc‐treated wild‐type or IL17R−/− mice; percentage of mice with at least one ANA. E denotes gestational day. ANA were determined with LIA at the age of 3 months


( A ) Time course of rapid internalization of α - synuclein fibrils by CADcells ( for characterization of α - synuclein assemblies , see Figure EV 1A ) . Internalization was measured by recording ATTO - 550 - positive neuron - like by flow cytometry . of ATTO - 550 - positive was quantified ( mean ± s . e . m ) ( left panel ) representative histograms of ATTO - 550 - positive are showed on right panel ( a . u . arbitrary units ) . Similarly , α - synuclein fibrils internalization was also confirmed by microscopy ( see Figure EV 1B ) .

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

(A) Time course of rapid internalization of fluorescent α-synuclein fibrils by CADcells (for characterization of α-synuclein assemblies, see Figure EV 1A). Internalization was measured by recording ATTO-550-positive neuron-like cells by flow cytometry. Percentage of ATTO-550-positive cells was quantified (mean ±s.e.m) (left panel) and representative histograms of ATTO-550-positive cells are showed on the right panel (a.u. arbitrary units). Similarly, α-synuclein fibrils internalization was also confirmed by fluorescent microscopy (see Figure EV 1B).


B ) Representative of donor ( upper panel ) acceptor ( lower panel ) after 24 h co - culture . Donor were loaded with α - synuclein fibrils prior to co - culture with GFP - transfected acceptor ; in red : α - synuclein fibrils , in green : acceptor in blue : nuclei . Scale bar represents 10 μm . ( n = 3 independent experiments ) . A larger field where donor acceptor are shown is presented in Figure EV 3A . ( C ) of donor acceptor containing α - synuclein fibrils after co - culture as in B : acceptor received α - synuclein fibrils . ( D ) of number of α - synuclein fibrils in donor acceptor after co - culture as in B . Donor contain around 70 α - synuclein fibrils ( median ) while acceptor contain 38 α - synuclein fibrils , respectively ( * * * * , p < 0 . 0001 by two - Mann Whitney ) . ( E ) of average of α - synuclein fibrillar foci in donor acceptor after co - culture as in B . ( * * * * , p < 0 . 0001 by two - Mann Whitney ) . In Figure EV 3 is shown an example of α - synuclein fibrillar detection in an acceptor . After detection , number of foci were determined using ICY software .

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

B) Representative images of donor (upper panel) and acceptor cells (lower panel) after 24 h co-culture. Donor cells were loaded with α-synuclein fibrils prior to co-culture with GFP-transfected acceptor cells; in red: α-synuclein fibrils, in green: acceptor cells and in blue: nuclei. Scale bar represents 10 μm. (n = 3 independent experiments). A larger field where donor and acceptor cells are shown is presented in Figure EV 3A.(C) Percentage of donor and acceptor cells containing α-synuclein fibrils after co-culture as in B: all acceptor cells received α-synuclein fibrils.(D) Quantification of the number of α-synuclein fibrils in donor and acceptor cells after co-culture as in B. Donor cells contain around 70 α-synuclein fibrils puncta (median) while acceptor cells contain 38 α-synuclein fibrils puncta, respectively (****, p < 0.0001 by two-tailed Mann Whitney test).(E) Quantification of the average size of α-synuclein fibrillar foci in donor and acceptor cells after co-culture as in B. (****, p < 0.0001 by two-tailed Mann Whitney test). In Figure EV 3 is shown an example of α-synuclein fibrillar puncta detection in an acceptor cell. After detection, the number and the size of foci were determined using the ICY software.


( A ) Representative confocal showing ChFP - α - syn transfected ( in red ) cultured alone ( upper ) or co - cultured with donor containing Alexa - 488 α - synuclein fibrils ( in green ) for 72 h ( bottom ) . arrow is pointing out representative α - synuclein fibrils Alexa - 488 co - localized with ChFP - α - syn . Scale bar represents 10 μm . Nuclei are stained with DAPI ( blue ) ( n = 3 independent experiments ) . ( B ) of number of ChFP - α - synuclein in ChFP - α - syn acceptor showed a significant increase in ChFP - α - synuclein number when were co - cultured with donor containing α - synuclein fibrils ( * * * , p < 0 . 001 by two - Mann Whitney ) . ( C ) of of co - localization of α - synuclein fibrils Alexa - 488 ChFP - α - syn . Data shows mean ± s . e . m . After co - culture , 11 % of transferred α - synuclein fibrils Alexa - 488 co - localized with ChFP - α - syn

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

(A) Representative confocal images showing ChFP-α-syn transfected cells (in red) cultured alone (upper) or co-cultured with donor cells containing fluorescent Alexa-488 α-synuclein fibrils (in green) for 72 h (bottom). The arrow is pointing out representative α-synuclein fibrils Alexa-488 co-localized with ChFP-α-syn puncta. Scale bar represents 10 μm. Nuclei are stained with DAPI (blue) (n=3 independent experiments).(B) Quantification of the number of ChFP-α-synuclein puncta in ChFP-α-syn acceptor cells showed a significant increase in ChFP-α-synuclein puncta number when cells were co-cultured with donor cells containing α-synuclein fibrils (***, p < 0.001 by two-tailed Mann Whitney test).(C) Quantification of the percentage of co-localization of α-synuclein fibrils Alexa-488 and ChFP-α-syn puncta. Data shows mean ± s.e.m. After co-culture, 11% of the transferred α-synuclein fibrils Alexa-488 co-localized with ChFP-α-syn puncta


( A ) Representative of donor ( upper panel ) acceptor ( bottom panel ) of co - culture system explained in Figure EV 2E ; in red : α - synuclein fibrils ATTO - 550 , in green : α - synuclein fibrils Alexa - 488 in blue : nuclei . Scale bar represents 10 μm ( n = 3 independent experiments ) . In insets , arrows point to co - localized ATTO - 550 Alexa - 488 α - synuclein fibrils whereas arrowheads point to of α - synuclein Alexa - 488 fibrils that do not co - localize with fibrillar ATTO - 550 α - synuclein .

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

(A) Representative images of donor cells (upper panel) and acceptor cells (bottom panel) of the co-culture system explained in Figure EV 2E; in red: α-synuclein fibrils ATTO-550, in green: α-synuclein fibrils Alexa-488 and in blue: nuclei. Scale bar represents 10 μm (n=3 independent experiments). In insets, arrows point to co-localized ATTO-550 and Alexa-488 α-synuclein fibrils puncta whereas arrowheads point to puncta of α-synuclein Alexa-488 fibrils that do not co-localize with fibrillar ATTO-550 α-synuclein.


( B ) of of Alexa - 488 α - synuclein fibrils co - localizing with α - ATTO - 550 α - synuclein fibrils reverse co - localization in discrete in donor acceptor as in A . Both measurements revealed high co - localization of two fluorophores in donor ( bar ) , but less in acceptor .

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

(B) Quantification of the percentage of Alexa-488 α-synuclein fibrils co-localizing with α- ATTO-550 α-synuclein fibrils and the reverse co-localization in discrete puncta in donor and acceptor cells as in A. Both measurements revealed high co-localization of the two fluorophores in donor cells (white bar), but less in acceptor cells.


( C ) amount of fibrils taken up by CAD exposed to 1µM fibrils , of fibrils remaining in exported into medium after 24h incubation were quantified by a filter trap . Data are mean ± s . d ( n = 3 independent measurements , filtered in duplicate ) . standard curve for increasing ATTO550 - α - synucleinfibrils concentrations is given .

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

(C) The amount of fibrils taken up by CAD cells exposed to 1µM fibrils, of fibrils remaining in the cells and exported into the medium after 24h incubation were quantified by a filter trap assay. Data are mean ± s.d (n=3 independent measurements, filtered in duplicate). The standard fluorescence curve for increasing ATTO550-α-synucleinfibrils concentrations is given.


( D ) Representative of GFP - transfected acceptor that were either ( i ) co - cultured with donor ( upper panel , Co - culture ) , ( ii ) cultured with conditioned medium of donor ( middle panel , CM ) or ( iii ) physically separated from donor using a filter ( bottom panel , Filter ) . Prior to culture , donor were loaded with ATTO - 550 α - synuclein fibrils . In red : α - synuclein fibrils , in green : acceptor in blue : nuclei . Scale bar represents 10 μm ( n = 3 independent experiments ) . ( E ) of of acceptor containing α - synuclein fibrils from such as those presented in C . When acceptor were cultured with conditioned medium from donor previously treated with α - synucleinfibrils ( not diluted , concentrated or diluted ) or co - cultured with a filter , of acceptor containing α - synuclein was low . Data shows mean ± s . e . m ( * * * , p < 0 . 001 ; by two - Student ' s t - ) . ( F ) of number of of α - synuclein fibrils per acceptor from D . While in co - culture condition number of α - synuclein in acceptor is on average 35 ( median ) , this number was on average 1 per acceptor in CM filter conditions , respectively ( * * * , p < 0 . 001 by two - Mann Whitney ) .

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

(D) Representative images of GFP-transfected acceptor cells that were either (i) co-cultured with donor cells (upper panel, Co-culture), (ii) cultured with the conditioned medium of donor cells (middle panel, CM) or (iii) physically separated from donor cells using a filter (bottom panel, Filter). Prior to culture, donor cells were loaded with ATTO-550 α-synuclein fibrils. In red: α-synuclein fibrils, in green: acceptor cells and in blue: nuclei. Scale bar represents 10 μm (n = 3 independent experiments).(E) Quantification of the percentage of acceptor cells containing α-synuclein fibrils from images such as those presented in C. When acceptor cells were cultured with the conditioned medium from donor cells previously treated with α-synucleinfibrils (not diluted, concentrated or diluted) or co-cultured with a filter, the percentage of acceptor cells containing α-synuclein puncta was low. Data shows mean ± s.e.m (***, p < 0.001; by two-tailed Student's t-test).(F) Quantification of the number of puncta of α-synuclein fibrils per acceptor cell from D. While in co-culture condition the number of α-synuclein puncta in acceptor cells is on average 35 (median), this number was on average 1 puncta per acceptor cells in CM and filter conditions, respectively (***, p < 0.001 by two-tailed Mann Whitney test).


( A ) Representative of control ( upper panel ) or treated with α - synucleinfibrilsATTO - 550 ( bottom panel ) for 16 hours ; in red : α - synucleinfibrils in green : WGAAlexa - 488 ( plasma dye marker ) , blue : DAPI . Scale bar represents 10 μm ( n = 3 independent experiments ) . ( B ) Relative of TNT - connected after α - synucleinfibrils treatment as in A shows an increase in TNT number upon fibrils treatment . Data are mean ± s . e . m ( * * * , p < 0 . 001 by paired , two - Student ' s t - ) .

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

(A) Representative images of control cells (upper panel) or cells treated with α-synucleinfibrilsATTO-550 (bottom panel) for 16 hours; in red: α-synucleinfibrils and in green: WGAAlexa-488 (plasma membrane dye marker), blue: DAPI. Scale bar represents 10 μm (n = 3 independent experiments).(B) Relative percentage of TNT-connected cells after α-synucleinfibrils treatment as in A shows an increase in TNT number upon fibrils treatment. Data are mean ± s.e.m (***, p < 0.001 by paired, two-tailed Student's t-test).


( C ) Confocal showing one TNT connecting two loaded with α - synucleinfibrilsATTO - 550 ( in red ) 16 hours after co - culture stained with WGAAlexa - 488 ( in blue ) . image represents a Z - projection of several middle stacks where TNT is located ( top panel ) . Insets show 3D reconstructions of TNT using ICY software ( middle panel : merge ; bottom panel : red channel ) . Several α - synucleinfibrils ( red ) are present in lumen of TNT ( WGAAlexa - 488 in ) . Scale bars represent 10 μm ( confocal ) 4 μm ( insets ) .

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

(C) Confocal images showing one TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 (in red) 16 hours after co-culture and stained with WGAAlexa-488 (in blue). The image represents a Z-projection of the several middle stacks where the TNT is located (top panel). Insets show 3D reconstructions of the TNT using ICY software (middle panel: merge; bottom panel: red channel). Several α-synucleinfibrils (red) are present in the lumen of the TNT (WGAAlexa-488 in white). Scale bars represent 10 μm (confocal images) and 4 μm (insets).


( D ) Confocal image showing TNT connecting a donor containing α - synucleinfibrils GFP transfected acceptor ( top panel ) strongly suggesting α - synucleinfibrils transfer from donor to acceptor . Insets show 3D reconstructions of TNT containing α - synucleinfibrils in lumen of TNT ; in red : α - synucleinfibrils , in green : GFP transfected acceptor in blue : WGAAlexa - 488 . Scale bars represent 10 μm 3 μm for insets .

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

(D) Confocal image showing TNT connecting a donor cell containing α-synucleinfibrils and GFP transfected acceptor cell (top panel) strongly suggesting α-synucleinfibrils transfer from donor to acceptor cell. Insets show 3D reconstructions of TNT containing α-synucleinfibrils in the lumen of the TNT; in red: α-synucleinfibrils, in green: GFP transfected acceptor cells in blue: WGAAlexa-488. Scale bars represent 10 μm and 3 μm for the insets.


( E ) Relative of TNTs in containing α - synucleinfibrils untransfected ( control ) or transfected with Myosin 10 ( Myosin 10 ) . Overexpression of Myosin 10 in donor population led to an increase in number of TNTs .

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

(E) Relative percentage of TNTs in cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Overexpression of Myosin 10 in the donor cell population led to an increase in the number of TNTs.


( F ) of average number of ATTO - 550α - synucleinfibrils per acceptor after 24 hours co - culture with donor containing α - synucleinfibrils untransfected ( control ) or transfected with Myosin 10 ( Myosin 10 ) . Note that Myosin 10 overexpression increases both transfer of DiIvesicles number of transferred α - synuclein in acceptor ; n = 3 independent experiments ( * * , p < 0 . 01 by two - Mann Whitney ) .

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

(F) Quantification of the average number of ATTO-550α-synucleinfibrils per acceptor cell after 24 hours co-culture with donor cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Note that Myosin 10 overexpression increases both the transfer of DiIvesicles and the number of transferred α-synuclein puncta in acceptor cells; n = 3 independent experiments (**, p < 0.01 by two-tailed Mann Whitney test).


( G ) of relative proportion of TNTs in co - culture of donor containing α - synuclein GFP acceptor under sub - confluent or sparse culture conditions . Data are mean ± s . e . m ; n = 3 independent experiments ( * , p < 0 . 05 by paired , two - Student ' s t - ) . ( H ) of of acceptor containing α - synuclein of co - culture in G . n = 3 independent experiments ( * , p < 0 . 05 by two - Mann Whitney ) . Condition impairing TNT formation ( i . e . , sparse condition ) induces a drastic decrease of TNT number α - synucleinfibrils transfer compared to condition allowing TNT formation ( sub - confluent condition ) .

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

(G) Quantification of the relative proportion of TNTs in co-culture of donor cells containing α-synuclein puncta and GFP acceptor cells under sub-confluent or sparse culture conditions. Data are mean ± s.e.m; n = 3 independent experiments (*, p < 0.05 by paired, two-tailed Student's t-test).(H) Quantification of the percentage of acceptor cells containing α-synuclein puncta of the co-culture in G. n = 3 independent experiments (*, p < 0.05 by two-tailed Mann Whitney test). Condition impairing TNT formation (i.e., sparse condition) induces a drastic decrease of TNT number and α-synucleinfibrils transfer compared to condition allowing TNT formation (sub-confluent condition).


( A ) Subcellular localization of α - synucleinfibrils in donor after 24 hours co - culture . left panels show representative of co - localization of α - synuclein with early endosomes ( EEA1 , top panel ) , recycling endosomes ( Vamp3 , middle panel ) lysosomal vesicles ( Lamp1 , bottom panel ) ( see arrows for co - localized spots ) ; in red : α - synucleinfibrils , in green : EEA1 , Vamp3 or Lamp1 in blue : nuclei . Scale bar represents 10 μm . On right , bar graph represents of α - synucleinpuncta co - localized with endo - lysosomal vesicles in A . Data are mean ± s . e . m . from three independent experiments .

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

(A) Subcellular localization of α-synucleinfibrils in donor cells after 24 hours co-culture. The left panels show representative images of co-localization of α-synuclein puncta with early endosomes (EEA1, top panel), recycling endosomes (Vamp3, middle panel) and lysosomal vesicles (Lamp1, bottom panel) (see arrows for co-localized spots); in red: α-synucleinfibrils, in green: EEA1, Vamp3 or Lamp1 and in blue: nuclei. Scale bar represents 10 μm. On the right, the bar graph represents the percentage of α-synucleinpuncta co-localized with endo-lysosomal vesicles in A. Data are mean ± s.e.m. from three independent experiments.


( B ) Confocal showing a TNT connecting two loaded with α - synucleinfibrilsATTO - 550 immunostained with Lamp1 ( in green ) . Labelling of TNTs is performed using a plasma dye ( WGAAlexa - 488 , in blue ) . image represents a Z - projection of several middle stacks where TNT is located . merged image with WGA - Alexa 633 ( blue ) , orthogonal view insets showing 3D reconstructions of TNT ( with WGA in ) are in right panel . Several α - synuclein fibrillar foci ( in red ) are present in lumen of TNT embedded in Lamp1 positive vesicles ( in green ) . Of note , only tubular structures physically connecting remote not touching substratum were identified as TNTs to distinguish them from filopodia . Scale bars represent 10 μm ( confocal ) 3 μm ( insets ) . merge of α - synucleinfibrils ( red ) Lamp1 ( green ) channels respective single channels are showed on left panel .

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

(B) Confocal images showing a TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 immunostained with Lamp1 (in green). Labelling of TNTs is performed using a plasma membrane dye (WGAAlexa-488, in blue). The image represents a Z-projection of the several middle stacks where the TNT is located. The merged image with WGA-Alexa 633 (blue), the orthogonal view and insets showing 3D reconstructions of the TNT (with WGA in white) are in the right panel. Several α-synuclein fibrillar foci (in red) are present in the lumen of the TNT and embedded in Lamp1 positive vesicles (in green). Of note, only tubular structures physically connecting remote cells and not touching the substratum were identified as TNTs to distinguish them from filopodia. Scale bars represent 10 μm (confocal images) and 3 μm (insets). The merge of α-synucleinfibrils (red) and Lamp1 (green) channels and the respective single channels are showed on the left panel.


( C ) top panels show representative of GFP - transfected acceptor after 24 h co - culture with donor containing α - synucleinfibrilsATTO - 550 . Immunofluorescence was performed with endo - lysosomal organelle markers ( top panel : EEA1 , middle panel : Vamp3 Lamp1 : bottom panel ) revealed that α - synucleinfibrils co - localized with lysosomal vesicles ( see arrow ) ; in red : α - synucleinfibrils , in green : organelle markers in blue : nuclei . Scale bar represents 10 μm , arrows pinpoint assemblies co - localizing with organelle markers . bar graph at bottom shows of of co - localization of α - synucleinfibrils with organelle markers , revealing that α - synuclein co - localized mostly with lysosomal vesicles . Data show mean ± s . e . m . from three independent experiments .

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

(C) The top panels show representative images of GFP-transfected acceptor cells after 24 h co-culture with donor cells containing α-synucleinfibrilsATTO-550. Immunofluorescence was performed with endo-lysosomal organelle markers (top panel: EEA1, middle panel: Vamp3 and Lamp1: bottom panel) and revealed that α-synucleinfibrils co-localized with lysosomal vesicles (see arrow); in red: α-synucleinfibrils, in green: organelle markers and in blue: nuclei. Scale bar represents 10 μm, arrows pinpoint assemblies co-localizing with organelle markers. The bar graph at the bottom shows the quantification of the percentage of co-localization of α-synucleinfibrils with organelle markers, revealing that α-synuclein puncta co-localized mostly with lysosomal vesicles. Data show mean ± s.e.m. from three independent experiments.


( D ) Representative confocal pictures of control loaded with ATTO - 550α - synucleinfibrils LysoTracker Deep Red ( top panel ) , of donor ( middle panel ) acceptor GFP - transfected acceptor ( bottom panel , GFP - vector not shown ) , loaded with α - synucleinfibrilsATTO - 550 LysoTracker Deep Red ( middle panel ) co - cultured for 24 hours ; in red : α - synucleinfibrils , in green : LysoTracker Deep Red in blue : nuclei . Scale bar represents 10 μm , arrows in inset pinpoint fibrils co - localizing with LysoTracker positive vesicles . ( E ) of co - localization of LysoTracker positive vesicles with α - synucleinfibrils revealed that 80 % of lysosomal vesicles that transferred from donor to acceptor contained α - synucleinfibrils thus demonstrating direct transfer of α - synucleinfibrils from donor to acceptor in majority inside lysosomal vesicles derived from donor . Data show mean ± s . e . m . from three independent experiments .

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

(D) Representative confocal pictures of control cells loaded with ATTO-550α-synucleinfibrils and LysoTracker Deep Red (top panel), and of donor (middle panel) and acceptor GFP-transfected acceptor cells (bottom panel, GFP-vector not shown) cells, loaded with α-synucleinfibrilsATTO-550 and LysoTracker Deep Red (middle panel) and co-cultured for 24 hours; in red: α-synucleinfibrils, in green: LysoTracker Deep Red and in blue: nuclei. Scale bar represents 10 μm, arrows in inset pinpoint fibrils co-localizing with LysoTracker positive vesicles.(E) The percentage of co-localization of LysoTracker positive vesicles with α-synucleinfibrils revealed that 80% of lysosomal vesicles that transferred from donor to acceptor cells contained α-synucleinfibrils thus demonstrating direct transfer of α-synucleinfibrils from donor to acceptor cells in majority inside lysosomal vesicles derived from donor cells. Data show mean ± s.e.m. from three independent experiments.


( A ) Representative of control ( upper panel ) α - synuclein loaded neurons ( middle , 0 . 5µM , bottom , 1µM , panels ) after 16 h . In red : α - synuclein fibrils , in green : MAP - 2 in blue : nuclei . Scale bar represents 10 μm .

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

(A) Representative images of control (upper panel) and α-synuclein loaded neurons (middle, 0.5µM, and bottom, 1µM, panels) after 16 h. In red: α-synuclein fibrils, in green: MAP-2 and in blue: nuclei. Scale bar represents 10 μm.


( B ) Representative of donor ( upper panels ) acceptor neurons ( bottom panels ) after 72 hours in co - culture . Donor neurons were loaded with α - synuclein fibrils prior to co - culture with CTG - labelled acceptor neurons then were labelled either with MAP - 2 ( first third panels ) or Lamp1 ( second fourth panels ) . In red : α - synuclein fibrils , in green : CTG , in : MAP - 2 / Lamp1 in blue : nuclei . Scale bar represents 5 μm . ( C ) graph bar shows of donor ( bar ) acceptor ( black bar ) neurons containing α - synuclein . Data show mean ± s . e . m . ( D ) box plot depicts number of in donor ( ) acceptor ( grey ) neurons . ( E ) Box plot showing distribution of average of α - synuclein in donor ( ) acceptor ( grey ) neurons . ( F ) graph bar represents of α - synuclein co - localized with lysosomes in donor ( ) acceptor ( black ) neurons . Data show mean ± s . e . m . Graphs in C , D , E F correspond to data from 3 independent experiments analysed after 72 hours of co - culture ( * * * * , p < 0 . 0001 compared to donor by two - Mann Whitney ) .

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

(B) Representative images of donor (upper panels) and acceptor neurons (bottom panels) after 72 hours in co-culture. Donor neurons were loaded with α-synuclein fibrils prior to co-culture with CTG-labelled acceptor neurons and then cells were labelled either with MAP-2 (first and third panels) or Lamp1 (second and fourth panels). In red: α-synuclein fibrils, in green: CTG, in white: MAP-2/ Lamp1 and in blue: nuclei. Scale bar represents 5 μm.(C) The graph bar shows the percentage of donor (white bar) and acceptor (black bar) neurons containing α-synuclein puncta. Data show mean ± s.e.m.(D) The box plot depicts the number of puncta in donor (white) and acceptor (grey) neurons.(E) Box plot showing the distribution of the average size of α-synuclein puncta in donor (white) and acceptor (grey) neurons.(F) The graph bar represents the percentage of α-synuclein puncta co-localized with lysosomes in donor (white) and acceptor (black) neurons. Data show mean ± s.e.m. Graphs in C, D, E and F correspond to data from 3 independent experiments analysed after 72 hours of co-culture (****, p < 0.0001 compared to donor by two-tailed Mann Whitney test).


( B ) Representative of acceptor neurons that were either ( i ) co - cultured with donor neurons ( upper panel , Co - culture ) , ( ii ) physically separated from donor ( middle panel , No contact ) or ( iii ) cultured with conditioned medium of donor neurons ( bottom panel , CM ) . In red : α - synucleinfibrils , in : acceptor neurons in blue : nuclei . 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O

(B) Representative images of acceptor neurons that were either (i) co-cultured with donor neurons (upper panel, Co-culture), (ii) physically separated from donor cells (middle panel, No contact) or (iii) cultured with the conditioned medium of donor neurons (bottom panel, CM). In red: α-synucleinfibrils, in white: acceptor neurons and in blue: nuclei. Scale bar represents 10 μm.


( C ) graph bar shows of acceptor containing α - synuclein from such as those presented in B . * * * * , p < 0 . 001 compared to co - culture condition by two - Student ' s t - . Data show mean ± s . e . m from 3 independent experiments . ( D ) box plot shows number of α - synuclein in acceptor neurons in co - culture ( ) , in CM ( grey ) in No contact condition ( grey ) . * * * * , p < 0 . 001 compared to co - culture condition by two - Mann Whitney ( n = 3 independent experiments ) . .

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

(C) The graph bar shows the percentage of acceptor cells containing α-synuclein puncta from images such as those presented in B. ****, p < 0.001 compared to the co-culture condition by two-tailed Student's t-test. Data show mean ± s.e.m from 3 independent experiments.(D) The box plot shows the number of α-synuclein puncta in acceptor neurons in co-culture (white), in CM (grey) and in the No contact condition (light grey). ****, p < 0.001 compared to the co-culture condition by two-tailed Mann Whitney test (n=3 independent experiments)..


( E ) Western blot of α - synuclein indicates presence of some α - synuclein in CM of neurons loaded with fibrils , after 72h compared to control medium to lysate of directly exposed to fibrils . α - tubulin was used as loading control .

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

(E) Western blot analysis of α-synuclein indicates the presence of some α-synuclein in the CM of neurons loaded with the fibrils, after 72h compared to control medium and to lysate of cells directly exposed to the fibrils. α-tubulin was used as loading control.


( A ) Representative of donor ( upper panel ) acceptor ( bottom panel ) after 24 hours in co - culture . Donor neurons were loaded with α - synucleinfibrils prior to co - culture with H2B - GFP - acceptor CAD . In red : α - synuclein , in green : acceptor CADs , in donor neurons in blue : nuclei . of donor are representative Z - stack projections of lower slices where these are located , corresponding to acceptor CADs are Z - stack projections of upper slices , covering whole . Scale bar represents 10 μm . ( B ) Box plots showing number ( left side ) average ( right side ) of α - synuclein in acceptor CAD after 24 hours in co - culture with donor neurons . ( n = 3 independent experiments ) .

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

(A) Representative images of donor (upper panel) and acceptor cells (bottom panel) after 24 hours in co-culture. Donor neurons were loaded with α-synucleinfibrils prior to co-culture with H2B-GFP-acceptor CAD cells. In red: α-synuclein puncta, in green: acceptor CADs, in white donor neurons and in blue: nuclei. The images of donor cells are representative Z-stack projections of the lower slices where these cells are located, and the images corresponding to acceptor CADs are Z-stack projections of the upper slices, covering the whole cell body. Scale bar represents 10 μm.(B) Box plots showing the number (left side) and average size (right side) of α-synuclein puncta in acceptor CAD cells after 24 hours in co-culture with donor neurons. (n=3 independent experiments).


( C ) Representative showing a TNT detected between CAD neurons after 24 hours in co - culture . In green : MAP - 2 , in : WGA , in red : α - synuclein in blue nuclei . merge image in bottom panel shows in detail TNT connecting , insets 3D reconstruction of neuron ( green ) CAD ( ) show presence of α - synuclein inside TNT .

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

(C) Representative images showing a TNT detected between CAD cells and neurons after 24 hours in co-culture. In green: MAP-2, in white: WGA, in red: α-synuclein puncta and in blue nuclei. The merge image in the bottom panel shows in detail the TNT connecting the cells, and the insets and the 3D reconstruction of the neuron (green) and the CAD cell (white) show the presence of α-synuclein puncta inside the TNT.


( C ) Asynchronous exponentially growing were treated with 0 . 2M HU ( + HU ) , then HU was removed from medium ( - HU ) . SDS - PAGE of total extracts taken at indicated times were used to detect kinetic of Rad53 phosphorylation upshift ( Rad53 - P = * * ) . Red green asterisk indicate time where mec1 - S1991 phosphomutants show altered Rad53 recovery . A 5 - fold dilution series of from exponential SC cultures of indicated strains were spotted on SC + / - indicated dose of HU .

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

(C) Asynchronous exponentially growing cells were treated with 0.2M HU (+HU), then HU was removed from the medium (-HU). SDS-PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift (Rad53-P=**). Red and green asterisk indicate the time where mec1-S1991 phosphomutants show altered Rad53 recovery. A 5-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of HU.


of replication fork progression at single - molecule level by combing . ( C ) Representative of . Green : ssDNA , red : BrdU . Scale bar corresponds to 20 kb . ( D ) Graph depict distribution of BrdU track . WT 90 min ( n = 427 ) 180 min ( n = 483 ) , mec1 - S1991A 90 min ( n = 309 ) 180 min ( n = 477 ) .

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

Analysis of replication fork progression at the single-molecule level by DNA combing. (C) Representative images of DNA fibers. Green: ssDNA, red: BrdU. Scale bar corresponds to 20 kb. (D) Graph depict the distribution of BrdU track length. WT 90 min (n=427) and 180 min (n=483), mec1-S1991A 90 min (n=309) and 180 min (n=477).


( A ) tRNA level measured by RT - qPCR in asynchronous ( async . ) culture after 90 min on HU in indicated strains . Expression is normalized to ACT1 . repression is expressed as a of HU treated / asynchronous in .

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

(A) tRNA level measured by RT-qPCR in asynchronous (async.) culture and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. The repression is expressed as a ratio of HU treated/asynchronous cells in percent.


( A - D ) Exponentially growing were treated with 0 . 2M HU total extracts were collected at indicated time points ( in min ) , samples were subjected to SDS - PAGE followed by immunoblotting with Rpb1 Tubulin antibodies . ( B , D ) Quantitation of total Rpb1 over time was done by normalizing Rpb1 to tubulin .

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

(A-D) Exponentially growing cells were treated with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies. (B,D) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin.