Immunoblots of BTV - NS3 - transfected 293T cells treated with proteasome inhibitors MG or LC . Membranes were probed for STAT1 , STAT2 , BTV - NS3 ( FLAG ) and GAPDH . ( B - D ) Immunoblots of BTV - NS3 - transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors ( B ) CQ or ( C ) NH4Cl or with increasing concentration of the ( D ) autophagosome formation inhibitor 3 - MA , and probed ( IB ) for STAT1 , STAT2 , BTV - NS3 ( FLAG ) and GAPDH expression .

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Immunoblots of BTV-NS3-transfected 293T cells treated with proteasome inhibitors MG or LC. Membranes were probed for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH. (B-D) Immunoblots of BTV-NS3-transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors (B) CQ or (C) NH4Cl or with increasing concentration of the (D) autophagosome formation inhibitor 3-MA, and probed (IB) for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH expression.


293T cells were co - transfected with NS3 expression plasmid ( or empty plasmid as control ) and siRNA for Atg7 Lysates were obtained from FACS - sorted transfected cells . Immunoblots of sorted cells were probed for STAT1 ; STAT2 ; Atg7 FLAG - tagged NS3 ; LC3 and GAPDH .

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293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Atg7 Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Atg7 FLAG-tagged NS3; LC3 and GAPDH.


293T cells were co - transfected with NS3 expression plasmid ( or empty plasmid as control ) and siRNA for Beclin - 1 ( or siRNA control or no RNA as controls ) . Lysates were obtained from FACS - sorted transfected cells . Immunoblots of sorted cells were probed for STAT1 ; STAT2 ; Beclin - 1 FLAG - tagged NS3 ; LC3 and GAPDH .

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293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Beclin-1 (or siRNA control or no RNA as controls). Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Beclin-1 FLAG-tagged NS3; LC3 and GAPDH.


Representative flow cytometry dot plots of Vero cells infected with rgBTV - 8 or BTV - 8 ΔNS3 and stained for BTV - VP7 . Gating on VP7 + cells was used for sorting to obtain lysates from infected cells .

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Representative flow cytometry dot plots of Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and stained for BTV-VP7. Gating on VP7+ cells was used for sorting to obtain lysates from infected cells.


Immunoblot of FACS - sorted Vero cells infected with rgBTV - 8 or BTV - 8 ΔNS3 and treated with IFN - U for 30min prior to fixation , staining and sorting . Membranes were probed for STAT1 , P - STAT1 , STAT2 and GAPDH .

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Immunoblot of FACS-sorted Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and treated with IFN-U for 30min prior to fixation, staining and sorting. Membranes were probed for STAT1, P-STAT1, STAT2 and GAPDH.


Immunofluorescence confocal images of Vero cells infected with rgBTV - 8 , BTV - 8 ΔNS3 or mock - infected and treated with IFN - U for 30min prior to fixation and staining . Cells were stained for BTV - VP7 and ( I ) P - STAT1 or ( J ) STAT2 . Nuclei were counterstained with DAPI . ( Scale bar = 30 μm ) . ( I ) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV - 8 ΔNS3 infected cells . ( J ) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV - 8 ΔNS3 - infected cells .

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Immunofluorescence confocal images of Vero cells infected with rgBTV-8, BTV-8 ΔNS3 or mock-infected and treated with IFN-U for 30min prior to fixation and staining. Cells were stained for BTV-VP7 and (I) P-STAT1 or (J) STAT2. Nuclei were counterstained with DAPI. (Scale bar = 30 μm). (I) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV-8 ΔNS3 infected cells. (J) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV-8 ΔNS3-infected cells.


WCE and FLAG - tag NS3 IP assay immunoblots of 293T transfected with BTV - NS3 - FLAG plasmids and increasing amounts of Ubiquitin - HA plasmids , and probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 ( FLAG ) or GAPDH expression .

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WCE and FLAG-tag NS3 IP assay immunoblots of 293T transfected with BTV-NS3-FLAG plasmids and increasing amounts of Ubiquitin-HA plasmids, and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG) or GAPDH expression.


Immunofluorescence staining of Vero cells co - transfected with BTV - NS3 and ubiquitin and acquired with Airyscan super - resolution . Scale bar = 10 μm . Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co - incidence ( scale bar = 2 μm ) .

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Immunofluorescence staining of Vero cells co-transfected with BTV-NS3 and ubiquitin and acquired with Airyscan super-resolution. Scale bar = 10 μm. Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co-incidence (scale bar = 2 μm).


Fluorescence intensity profile of cell section indicated by the white line ( blue : DAPI , green : NS3 ; red : Ub ) . Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub .

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Fluorescence intensity profile of cell section indicated by the white line (blue: DAPI, green: NS3; red: Ub). Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub.


FLAG - tag IP assay of BTV - NS3 and WCE immunoblot of 293T co - transfected with BTV FLAG - NS3 - , - NS3 - K13R , - NS3 - K15R , - NS3 - K13 / 15R or - NS3 - PPRY / AARH plasmid and Ubiquitin - HA plasmid and probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 ( FLAG ) , STAT2 and GAPDH expression .

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FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected with BTV FLAG-NS3-, -NS3-K13R, -NS3-K15R, -NS3-K13/15R or -NS3-PPRY/AARH plasmid and Ubiquitin-HA plasmid and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG), STAT2 and GAPDH expression.


WCE and HA - tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin - HA plasmids and increasing amounts of BTV - FLAG - NS3 - K13 / 15R , and probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 - K13 / 15R ( FLAG ) or GAPDH expression .

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WCE and HA-tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin-HA plasmids and increasing amounts of BTV-FLAG-NS3-K13/15R, and probed (IB) for ubiquitin (HA), BTV-NS3-K13/15R (FLAG) or GAPDH expression.


ISRE - driven luciferase reporter assay in 293T cells transfected with empty expression plasmid ( Control ) , BTV - NS2 ( 300 ng ) , - NS3 ( 50 , 300 and 600 ng ) , or - NS3 - K13 / 15R ( 50 , 300 and 600 ng ) - expressing plasmids . Mean % inductions of luciferase activity of three independent experiments are shown .

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ISRE-driven luciferase reporter assay in 293T cells transfected with empty expression plasmid (Control), BTV -NS2 (300 ng), -NS3 (50, 300 and 600 ng), or -NS3-K13/15R (50, 300 and 600 ng)-expressing plasmids. Mean % inductions of luciferase activity of three independent experiments are shown.


FLAG - tag IP assay of BTV - NS3 and WCE immunoblot of 293T co - transfected FLAG - BTV - NS3 plasmid and HA - UB - WT or HA - UB lysine ( K to R ) mutants . IP were probed ( IB ) for ubiquitin ( HA ) and BTV - NS3 ( FLAG ) expression . WCE were probed for STAT1 , STAT2 , ubiquitin ( HA ) , BTV - NS3 ( FLAG ) , and GAPDH expression .

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FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected FLAG-BTV-NS3 plasmid and HA-UB-WT or HA-UB lysine (K to R) mutants. IP were probed (IB) for ubiquitin (HA) and BTV-NS3 (FLAG) expression. WCE were probed for STAT1, STAT2, ubiquitin (HA), BTV-NS3 (FLAG), and GAPDH expression.


( a , b ) Bacteria in the spleens of Slamf1 − / − Rag1 − / − and Rag1 − / − BALB / c mice ( a ) or Slamf1 − / − and Slamf1 + / + BALB / c mice ( b ) 48 h after intraperitoneal injection of virulent S . typhimurium 14028s or attenuated S . typhimurium SseB − . CFU , colony - forming units ; ND , not detectable . Data are representative of four independent experiments ( mean and s . d . ) .

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(a,b) Bacteria in the spleens of Slamf1−/−Rag1−/− and Rag1−/− BALB/c mice (a) or Slamf1−/− and Slamf1+/+ BALB/c mice (b) 48 h after intraperitoneal injection of virulent S. typhimurium 14028s or attenuated S. typhimurium SseB−. CFU, colony-forming units; ND, not detectable. Data are representative of four independent experiments (mean and s.d.).


( c - e ) Killing of bacteria by peritoneal macrophages from Slamf1 + / + and Slamf1 − / − BALB / c mice exposed to S . typhimurium SseB − ( c ) , E . coli F18 ( d ) or S . aureus ( e ) , assessed by gentamycin assay . Data are representative of five independent experiments ( mean and s . d . ) .

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(c-e) Killing of bacteria by peritoneal macrophages from Slamf1+/+ and Slamf1−/− BALB/c mice exposed to S. typhimurium SseB− (c), E. coli F18 (d) or S. aureus (e), assessed by gentamycin assay. Data are representative of five independent experiments (mean and s.d.).


( f ) Uptake of bacteria by Slamf1 + / + ( red solid lines ) or Slamf1 − / − ( black solid lines ) BALB / c peritoneal macrophages incubated at 37 ° C with E . coli - eGFP or S . typhimurium - eGFP or by Slamf1 + / + macrophages incubated for 60 min at 4 ° C with the bacteria ( dotted lines ) . Right , mean fluorescence intensity ( MFI ) . Data are representative of three independent experiments ( mean and s . d . ) .

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(f) Uptake of bacteria by Slamf1+/+ (red solid lines) or Slamf1−/− (black solid lines) BALB/c peritoneal macrophages incubated at 37 °C with E. coli-eGFP or S. typhimurium-eGFP or by Slamf1+/+ macrophages incubated for 60 min at 4 °C with the bacteria (dotted lines). Right, mean fluorescence intensity (MFI). Data are representative of three independent experiments (mean and s.d.).


( a ) NOX2 activity in Slamf1 + / + and Slamf1 - / - BALB / c peritoneal macrophages stimulated for 0 - 100 min with E . coli F18 , S . aureus or PMA , assessed with lucigenin . Data are representative of five independent experiments ( mean ± s . d . ) .

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(a) NOX2 activity in Slamf1+/+ and Slamf1-/- BALB/c peritoneal macrophages stimulated for 0-100 min with E. coli F18, S. aureus or PMA, assessed with lucigenin. Data are representative of five independent experiments (mean ± s.d.).


( b ) Phagosomal pH of Slamf1 + / + , Slamf1 - / - and gp91phox - / - B6 primary macrophages loaded for 0 - 200 min with pHrodo - coated E . coli or S . aureus , analyzed by flow cytometry . Data are representative of three independent experiments ( mean ± s . d . ) .

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(b) Phagosomal pH of Slamf1+/+, Slamf1-/- and gp91phox-/- B6 primary macrophages loaded for 0-200 min with pHrodo-coated E. coli or S. aureus, analyzed by flow cytometry. Data are representative of three independent experiments (mean ± s.d.).


( c ) NOX2 activity in primary macrophages in response to LPS , purified OmpC , peptidoglycan ( PGN ) or PMA , assessed with lucigenin . TLR4 - KO , TLR4 - deficient ( strain del / Jtht ; C3H ) . Data are representative of five independent experiments ( mean ± s . d . ) .

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(c) NOX2 activity in primary macrophages in response to LPS, purified OmpC, peptidoglycan (PGN) or PMA, assessed with lucigenin. TLR4-KO, TLR4-deficient (strain del/Jtht; C3H). Data are representative of five independent experiments (mean ± s.d.).


( 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 .

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(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 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 .

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(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 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 . ) .

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(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 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 . ) .

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(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 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 . ) .

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(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 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 .

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(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 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 ) .

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(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 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 .

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(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 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 ) .

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(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 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 . ) .

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(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 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 .

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(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 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 .

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(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 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 . ) .

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(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 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 .

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(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 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 .

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(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 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 .

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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 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 .

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

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


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 .

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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 and Rod and Bub1 co - depleted HeLa , U2OS and RPE1 cells . Cells were treated with nocodazole .

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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 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 .

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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 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 .

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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.


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 .

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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 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 .

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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 cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped .

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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 cells treated with taxol . Red circles represent cells still arrested in mitosis when the filming stopped .

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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 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 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 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 cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.


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 .

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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 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 .

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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 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 .

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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 and the time from NEBD to exit was measured in each condition . Red circles represent cells still arrested in mitosis when the filming stopped .

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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 and the time from NEBD to exit was measured in each condition . Red circles represent cells still arrested in mitosis when the filming stopped .

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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 cells , Bub1 CR cells and Rod CR cells in the presence of nocodazole . Scale bar , 5 μm .

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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 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 .

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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 .

Analysis of Rod levels in cells used for FRAP experiment.


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 .

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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 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 .

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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.


The 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 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 .

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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.


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 .

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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.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells .

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


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 .

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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 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

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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 primary neural progenitors treated with CRH with or without antalarmin ( Ant ) ( n = 3 biological replicates ) .

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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 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 ) .

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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 the ( C ) neurosphere

Effects of CRHR1 overexpression on the (C) neurosphere formation


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

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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 ) .

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


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

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


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

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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 the ( H ) neurosphere morphology , n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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

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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 cells transduced with a lentiviral vector expressing shCRHR1 .

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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 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 ) .

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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 , and samples were harvested at E17 . 5 for anti - GFP immunolabeling . GFP + cell localization was quantified ( bottom panel ) ( n = 5 biological replicates ) .

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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 and co - immunostained using anti - GFP ( green ) together with ( E ) anti - TBR2 ( red ) or ( F ) anti - CTIP2 ( red ) antibodies . n = 3 biological replicates .

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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 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 ) .

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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).


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 .

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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.


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 .

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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.


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


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

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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 ) and SOX2 ( red ) antibodies .

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

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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 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 )

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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 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 .

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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 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 ) .

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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 ) 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 ' ' .

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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 ) and SOX2 ( red ) antibodies .

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

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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 cells transduced with CREB - expressing lentiviral vectors , measuring REST

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(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 cells transduced with CREB - expressing lentiviral vectors , measuring REST protein levels , respectively .

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(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 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 )

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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 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

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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 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 )

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 + 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 .

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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 − / − , LIGHT − / − , and Ltbr − / − Hox11 − / − mice ; percentage of mice with at least one ANA .

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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 individual mice .

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


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

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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 ; 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 .

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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 cell ‐ specific LT knockout mice ; percentage 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 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 .

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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 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 .

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

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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 and tested for ANA ; percentage of mice with at least one ANA reactivity .

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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 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 .

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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 , 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 .

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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 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 .

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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 ; percentage of mice with at least one ANA . E denotes gestational day . ANA were determined with LIA at the age of 3

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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 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 ) .

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(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 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 .

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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 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

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(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 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 .

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(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 ) 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 .

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(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 ) 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 .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 ) 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 ) .

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(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 ) 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 ) .

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(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 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 .

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(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.