( G - J ) Quantitative real - time polymerase chain reaction ( qPCR ) of miR - 1a ( G ) , miR - 9 ( H ) , miR - 146a ( I ) , miR - 155 ( J ) . Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR ( 0 h , n = 4 ; 1 h , n = 5 ; 6 h , n = 5 ; 24 h , n = 3 ) .

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(G-J) Quantitative real-time polymerase chain reaction (qPCR) analysis of miR-1a (G), miR-9 (H), miR-146a (I), and miR-155 (J). Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay (0 h, n=4; 1 h, n=5; 6 h, n=5; 24 h, n=3).


( K ) NanoSight of CSF isolated from LPS - injected mice followed by icv injection of vehicle or GW4869 , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 8 ) .

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(K) NanoSight analysis of CSF isolated from LPS-injected mice followed by icv injection of vehicle or GW4869, a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 8).


( L ) qPCR of expression of miR - 1a , miR - 9 , miR - 146a miR - 155 in choroid plexus of mice injected with LPS then icv injected with vehicle ( black ) or GW4869 ( grey ) , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 4 ) .

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(L) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle (black) or GW4869 (grey), a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 4).


( M ) NanoSight of supernatant of choroid plexus explants from PBS - or LPS - injected mice ( n = 6 ) . Data are displayed as mean ± analyzed by Student ' s t - . Significance are indicated on graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

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(M) NanoSight analysis of supernatant of choroid plexus explants from PBS- or LPS-injected mice (n = 6). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.


( A - B ) Representative confocal of choroid plexus ( CP ) on brain sections from naive mice four hours after LPS injection ( n = 3 ) . Brain sections were stained for TLR4 ( A ) TNFR1 ( B ) ( red ) , pan - cytokeratine ( panCK ; green ) nuclei were stained with Hoechst ( blue ) . ependymal aligning ventricles are marked with a dotted . Scalebar = 100 µm .

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(A-B) Representative confocal images of choroid plexus (CP) on brain sections from naive mice and four hours after LPS injection (n=3). Brain sections were stained for TLR4 (A) and TNFR1 (B) (red), pan-cytokeratine (panCK; green) and the nuclei were stained with Hoechst (blue). The ependymal cells aligning the ventricles are marked with a dotted line. Scalebar = 100 µm.


( C ) NanoSight of EVs in CSF in sham - operated ( black ) relative to CLP - treated ( grey ) mice 10 hours after surgery .

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(C) NanoSight analysis of EVs in the CSF in sham-operated (black) relative to CLP-treated (grey) mice 10 hours after surgery.


( D ) qPCR of expression of miR - 1a , miR - 9 , miR - 146a miR - 155 in choroid plexus of sham - operated mice ( black ) mice subjected to CLP ( grey ) 10 hours after surgery . ( E ) qPCR of expression of miR - 1a , miR - 9 , miR - 146a miR - 155 in CSF of sham - operated mice ( black ) mice subjected to CLP ( grey ) 10 hours after surgery .

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(D) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery. (E) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery.


( F ) NanoSight of EVs in CSF of control ( black ) TNF - injected ( 25 µg / 20 g ; grey ) mice 6 hours after TNF challenge .

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(F) NanoSight analysis of EVs in CSF of control (black) and TNF-injected (25 µg/20 g; grey) mice 6 hours after TNF challenge.


( G ) qPCR of expression of miR - 1a , miR - 9 , miR - 146a miR - 155 in choroid plexus of control mice ( black ) in mice injected with TNF ( grey ) 6 hours after injection . ( H ) qPCR of expression of miR - 1a , miR - 9 , miR - 146a miR - 155 in CSF of control mice ( black ) on mice 6 hours after TNF injection ( grey ) . Data are displayed as mean ± analyzed by Student ' s t - . Scalebar 100 µm . Significance are indicated on graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

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(G) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of control mice (black) and in mice injected with TNF (grey) 6 hours after injection. (H) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of control mice (black) and on mice 6 hours after TNF injection (grey). Data are displayed as mean ± SEM and analyzed by Student's t-test. Scalebar 100 µm. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.


( A ) Representative confocal image of brain parenchyma , four hours after intracerebroventricular injection of PKH26 labelled EVs ( red ) . Astrocytes are stained with GFAP ( ) , nuclei with Hoechst ( blue ) , pan - cytokeratin ( panCK ; green ) was used to label choroid plexus . dotted shows ventricular border arrow heads point to EVs that crossed ependymal layer .

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(A) Representative confocal image of brain parenchyma, four hours after intracerebroventricular injection of PKH26 labelled EVs (red). Astrocytes are stained with GFAP (white), nuclei with Hoechst (blue), and pan-cytokeratin (panCK; green) was used to label the choroid plexus. The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer.


( B - C ) Representative confocal showing uptake of double - labelled EVs ( Ribogreen ; green , PKH26 , red ) by astrocytes stained with GFAP ( ) ( B ) microglia stained with IBA1 ( ) ( C ) incubated on mixed cortical cultures . Boxed areas are shown as zoomed on right . nuclei are stained with Hoechst ( blue ) . in vivo in vitro uptake experiments were performed 3 2 times ( n = 3 ) , respectively . Scalebar 100 = µm .

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(B-C) Representative confocal images showing the uptake of double-labelled EVs (Ribogreen; green, PKH26, red) by astrocytes stained with GFAP (white) (B) and microglia cells stained with IBA1 (white) (C) incubated on mixed cortical cultures. Boxed areas are shown as zoomed images on right. Cell nuclei are stained with Hoechst (blue). The in vivo and in vitro uptake experiments were performed 3 and 2 times (n=3), respectively. Scalebar 100 = µm.


( A - B ) qPCR gene expression of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice ( A ) in brain tissue ( B ) before ( black ) 8 h after LPS injection ( grey ) ( n = 3 ) .

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(A-B) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice (A) and in brain tissue (B) before (black) and 8 h after LPS injection (grey) (n=3).


( C ) qPCR gene expression of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos iκbα by qPCR in mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice in brain tissue ( right ; in vivo ) before ( black ) 8 h after LPS injection ( grey ) ( n = 3 ) .

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(C) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα by qPCR in mixed cortical cultures (left; in vitro) incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice and in brain tissue (right; in vivo) before (black) and 8 h after LPS injection (grey) (n=3).


( D ) Cytokine ( IL - 6 , IL - 1β TNF ) of supernatant of mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from untreated ( black ; n = 3 ) or LPS treated ( grey ; n = 3 ) mice of CSF ( right ; in vivo ) from untreated ( black ; n = 3 ) LPS treated mice ( grey ; n = 6 ) .

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(D) Cytokine analysis (IL-6, IL-1β and TNF) of supernatant of mixed cortical cultures (left; in vitro) incubated with EVs isolated from untreated (black; n=3) or LPS treated (grey; n=3) mice and of CSF (right; in vivo) from untreated (black; n=3) and LPS treated mice (grey; n=6).


( E - F ) qPCR gene expression of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos iκbα ( E ) miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 Irak1 ( F ) in mixed cortical cultures incubated with CD63 - depleted EVs isolated from untreated mice ( black ) mice treated with LPS for 6 h ( grey ) ( n = 3 ) .

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(E-F) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (E) and miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (F) in mixed cortical cultures incubated with CD63-depleted EVs isolated from untreated mice (black) and mice treated with LPS for 6 h (grey) (n=3).


( G - H ) qPCR gene expression of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 Irak1 ( G ) inflammatory genes Il - 1β , Tnf , Il - 6 , iNos iκbα ( H ) in brain tissue from LPS - injected mice icv injected with vehicle ( black ) or GW4869 ( grey ; n = 7 ) . Data are displayed as mean ± analyzed by Student ' s t - . Significance are indicated on graphs : * , 0 . 01 ≤ P < 0 . 05 .

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(G-H) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (G) and inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (H) in brain tissue from LPS-injected mice icv injected with vehicle (black) or GW4869 (grey; n=7). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05.


A ) Double in situ hybridization using Nrg3 ( green ) Gad1 ( red ) specific probes ; DAPI was used as a counter stain . Nrg3 is present in Gad1 - positive ( filled arrowheads ) - negative neurons . ( A ' ) ( A ' ' ) show higher magnifications of boxed areas indicated in ( A ) . Nrg3 - negative neurons are indicated by open arrowheads ; note that many but not neurons express Nrg3 .

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A) Double fluorescence in situ hybridization using Nrg3 (green) and Gad1 (red) specific probes; DAPI was used as a counter stain. Nrg3 is present in Gad1-positive (filled arrowheads) and -negative neurons. (A') and (A'') show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3.


B ) Comparison of mRNA of Nrg3 , Nrg1β type III ( Nrg1β − III ) , Nrg2β , Nrg1β type II ( Nrg1β − II ) , Nrg2α Nrg1β type I ( Nrg1β − I ) in cortex ( Cx ) , hippocampus ( HC ) striatum ( Str ) of juvenile mice ( P30 - 60 ) by quantitative RT - PCR . Data represent means ± SD of 4 biological replicates . 2 - way - ANOVA with Bonferroni ' s multiple comparisons was performed to assess statistical significance ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , ns = not significant ) .

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B) Comparison of mRNA levels of Nrg3, Nrg1β type III (Nrg1β−III), Nrg2β, Nrg1β type II (Nrg1β−II), Nrg2α and Nrg1β type I (Nrg1β−I) in the cortex (Cx), hippocampus (HC) and striatum (Str) of juvenile mice (P30-60) by quantitative RT-PCR. Data represent means ± SD of 4 biological replicates. 2-way-ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (****P<0.0001, ***P<0.001, ns=not significant).


D ) Detection of Nrg3 by western blot in fractions from brain lysates : S1 , crude lysate ; S2 , cytosol membranes ; S2 ' , cytosol ; LM , membranes ; P2 ' , crude synaptosomes ; S3 ' , synaptosomal cytoplasm ; SV , synaptic vesicles ; P3 , synaptosomal membranes ; SPM , synaptic plasma membranes .

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D) Detection of Nrg3 by western blot analysis in fractions from brain lysates: S1, crude lysate; S2, cytosol and light membranes; S2', cytosol; LM, light membranes; P2', crude synaptosomes; S3', synaptosomal cytoplasm; SV, synaptic vesicles; P3, synaptosomal membranes; SPM, synaptic plasma membranes.


E , F ) Immunohistochemical in hippocampus of wildtype ( E , E ' , E ' ' ) ErbB4 mutant mice ( F ) at 2 month of using Nrg3 - , ErbB4 - parvalbumin ( Parv ) - specific antibodies . box in ( E ) is displayed magnified in ( E ' , E ' ' ) . In control mice , Nrg3 is enriched on dendrites of ErbB4 + PV interneurons , compared to surrounding neuropil ( E ' , E ' ' ) . In ErbB4 mutants , Nrg3 enrichment on PV interneurons is not apparent ( F ) .

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E, F) Immunohistochemical analysis in the hippocampus of wildtype (E,E',E'') and ErbB4 mutant mice (F) at 2 month of age using Nrg3-, ErbB4- and parvalbumin (Parv)-specific antibodies. The white box in (E) is displayed magnified in (E',E''). In control mice, Nrg3 is enriched on dendrites of ErbB4+ PV interneurons, compared to the surrounding neuropil (E',E''). In ErbB4 mutants, the Nrg3 enrichment on PV interneurons is not apparent (F).


G ) Nrg3 , ErbB4 GluA4 were identified by immunohistology co - localized in synaptic on dendrites of interneurons in stratum radiatum of hippocampal CA1 . Note that ( G ) displays Nrg3 / ErbB4 , ( G ' ) Nrg3 / GluA4 , ( G ' ' ) ErbB4 / GluA4 signals of same triple - stained image ; false colors were assigned for better signal visualization .

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G) Nrg3, ErbB4 and GluA4 were identified by immunohistology and co-localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1. Note that (G) displays Nrg3/ErbB4, (G') Nrg3/GluA4, and (G'') ErbB4/GluA4 signals of the same triple-stained image; false colors were assigned for better signal visualization.


H ) of GluA4 present on ErbB4 + dendrites in CA1 stratum radiatum of adult wildtype ( wt ) Nrg3 - / - mice ( P90 - 120 ) . Data are presented as box plots with Tukey ' s whiskers outliers ; means are indicated by a Plus symbols . n = 65 ( wt ) n = 62 ( Nrg3 - / - ) from 5 each . Unpaired t - ( 2 - ) with Welch ' s correction was performed to assess statistical significance ( * * * * P < 0 . 0001 ) .

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H) Quantification of GluA4 puncta present on ErbB4+ dendrites in the CA1 stratum radiatum of adult wildtype (wt) and Nrg3-/- mice (age P90-120). Data are presented as box plots with Tukey's whiskers and outliers; means are indicated by a Plus symbols. n=65 (wt) and n=62 (Nrg3-/-) from 5 animals each. Unpaired t-test (2-tailed) with Welch's correction was performed to assess statistical significance (****P<0.0001).


A ) His6 - tagged EGF domains of Nrg1β Nrg3 were added to neuronal cultures at indicated concentrations . Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho - tyrosine ( pY ) ErbB4 , of whole lysates using antibodies against pErk1 / 2 pAkt

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A) His6-tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho-tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt


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C , D ) Neurons from Nrg3 - / - ; ErbB4 - / - ; vGAT - Cre mice were transduced with a lentivirus expressing either ( C ) ErbB4wt or ( D ) ErbB4kd ( kinase dead ErbB4 ) in a Cre - dependent manner , with a second at a low titer expressing Nrg3 / SypCFP . Immunocytochemical of ErbB4 SypCFP ; ( C , C ' ) ( D , D ' ) each show same image , ( C ' , D ' ) show SypCFP signal

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C, D) Neurons from Nrg3-/-;ErbB4-/-;vGAT-Cre mice were transduced with a lentivirus expressing either (C) ErbB4wt or (D) ErbB4kd (kinase dead ErbB4) in a Cre-dependent manner, and with a second virus at a low titer expressing Nrg3/SypCFP. Immunocytochemical analysis of ErbB4 and SypCFP; (C,C') and (D,D') each show the same image, (C',D') show the SypCFP signal


E - G ) of Synapse preference towards ErbB4 + neurons ( E ) , ErbB4 enrichment ( F ) SypCFP enrichment ( G ) in synapses on neurons expressing wildtype ErbB4 ( wt ) or ErbB4kd ( kd )

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E-G) Quantification of Synapse preference towards ErbB4+ neurons (E), ErbB4 enrichment (F) and SypCFP enrichment (G) in synapses on neurons expressing wildtype ErbB4 (wt) or ErbB4kd (kd)


H ) Nrg3 - / - neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 GFP immunostained with antibodies against ErbB4 , GFP GluA ; ( H ) shows triple stained image , while ( H ' ) ( H ' ' ) only show ErbB4 GluA signals , respectively

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H) Nrg3-/- neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4, GFP and GluA; (H) shows the triple stained image, while (H') and (H'') only show ErbB4 and GluA signals, respectively


I ) Co - cultures of Nrg3 - / - neurons with HEK293 expressing Nrg3 . Cultures were stained with antibodies against Nrg3 , ErbB4 GluA . Note that Nrg3 , ErbB4 GluA are enriched at contact sites between Nrg3 - expressing HEK293 ErbB4 + neurons

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I) Co-cultures of Nrg3-/- neurons with HEK293 cells expressing Nrg3. Cultures were stained with antibodies against Nrg3, ErbB4 and GluA. Note that Nrg3, ErbB4 and GluA are enriched at the contact sites between Nrg3-expressing HEK293 cells and ErbB4+ neurons


C ) Example traces of evoked excitatory postsynaptic currents ( eEPSCs ) averaged over 50 sweeps . D , E ) of peak amplitude of first eEPSC ( D ) of paired ( E )

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C) Example traces of evoked excitatory postsynaptic currents (eEPSCs) averaged over 50 sweeps. D, E) Quantification of the peak amplitude of the first eEPSC (D) and of the paired pulse ratio (E)


F - I ) sEPSC recordings from CA1 pyramidal ( F , H ) PV neurons ( G , I ) in acute slices . sEPSC frequencies ( F , G ) amplitudes ( H , I ) are

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F-I) sEPSC recordings from CA1 pyramidal (F,H) and PV neurons (G, I) in acute slices. sEPSC frequencies (F,G) and amplitudes (H,I) are shown


TRF1 immunofluorescence of lungs . Notice absence presence of TRF1 signal in carcinomas surrounding healthy tissue of Trf1Δ / Δ mice , respectively .

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TRF1 immunofluorescence of the lungs. Notice the absence and presence of TRF1 signal in the carcinomas and surrounding healthy tissue of Trf1Δ/Δ mice, respectively.


of Trf1 excision by PCR . Notice completed excision in carcinomas of Trf1lox / lox lungs .

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Analysis of Trf1 excision by PCR. Notice the completed excision in carcinomas of Trf1lox/lox lungs.


Detection of β - galactosidase activity in lungs as a surrogate marker of oncogenic K - RasG12V expression .

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Detection of β-galactosidase activity in the lungs as a surrogate marker of oncogenic K-RasG12V expression.


growth curve of Trf1 + / + K - Ras + / G12Vp53 + / + Trf1Δ / ΔK - Ras + / G12Vp53 + / + measured by computed tomography ( CT ) . of number of Trf1 + / + K - Ras + / G12Vp53 + / + Trf1Δ / ΔK - Ras + / G12Vp53 + / + carcinomas at point .

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Tumor growth curve of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+ measured by computed tomography (CT).Quantification of the number and size of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+carcinomas at death point.


of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed . Post - mortem of Trf1 deletion in each revealed that none of Trf1lox / lox K - Ras + / G12V p53 + / + ones had excised Trf1 .

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Percentage of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed. Post-mortem analysis of Trf1 deletion in each tumor revealed that none of the Trf1lox/lox K-Ras+/G12V p53+/+ ones had excised Trf1.


growth curve growth slope of Trf1 + / + K - Ras + / G12V p53 − / − Trf1 − / − K - Ras + / G12V p53 − / − measured by CT .

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Tumor growth curve and tumor growth slope of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− measured by CT.


maximal section of Trf1 + / + K - Ras + / G12Vp53 − / − Trf1Δ / ΔK - Ras + / G12Vp53 − / − lungs measured by histological before point by CT .

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Tumor maximal section of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs measured by histological analysis before death point by CT.


Maximum 18F - FDG - glucose uptake by Trf1 + / + K - Ras + / G12Vp53 − / − Trf1Δ / ΔK - Ras + / G12Vp53 − / − tumors 22 after infection by positron emission tomography ( PET ) . Representative PET - CT image of Trf1 + / + K - Ras + / G12Vp53 − / − Trf1Δ / ΔK - Ras + / G12Vp53 − / − lungs .

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Maximum 18F-FDG-glucose uptake by Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−tumors 22 weeks after infection by positron emission tomography (PET).Representative PET-CT image of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs.


Survival curve of Trf1 + / + K - Ras + / G12V p53 − / − Trf1 − / − K - Ras + / G12V p53 − / − mice .

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Survival curve of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− mice.


of showing γH2AX foci in carcinomas of indicated genotypes ( left panel ) . Representative of γH2AX immunohistochemistry ( right panel ) .

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Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes (left panel). Representative images of γH2AX immunohistochemistry (right panel).


of showing 3 or more γH2AX RAP1 colocalizing foci ( TIFs ) ( left panel ) . Representative of γH2AX RAP1 double immunofluorescence ( right panel ) . Yellow arrowheads : colocalization of γH2AX RAP1 .

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Percentage of cells showing 3 or more γH2AX and RAP1 colocalizing foci (TIFs) (left panel). Representative images of γH2AX and RAP1 double immunofluorescence (right panel). Yellow arrowheads: colocalization of γH2AX and RAP1.


of active caspase - 3 ( AC3 ) - positive ( left panel ) . Representative of AC3 immunohistochemistry ( right panel ) .

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Percentage of active caspase-3 (AC3)-positive cells (left panel). Representative images of AC3 immunohistochemistry (right panel).


of Ki67 - positive in carcinomas of indicated genotypes ( left panel ) . Representative of Ki67 immunohistochemistry ( right panel ) .

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Percentage of Ki67-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of Ki67 immunohistochemistry (right panel).


of pH3 - positive in carcinomas of indicated genotypes ( left panel ) . Representative of pH3 immunohistochemistry ( right panel ) . Red arrowheads : pH3 - positive .

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Percentage of pH3-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of pH3 immunohistochemistry (right panel). Red arrowheads: pH3-positive cells.


of giant nuclei in carcinomas of indicated genotype . of anaphase bridges out of total anaphases in carcinomas of indicated genotypes . Representative of giant nuclei , multilobulated nuclei , anaphase bridges , multipolar mitoses . Red arrowheads indicate corresponding mitotic aberrations indicated in image .

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Percentage of giant nuclei in the carcinomas of the indicated genotype.Percentage of anaphase bridges out of total anaphases in the carcinomas of the indicated genotypes.Representative images of giant nuclei, multilobulated nuclei, anaphase bridges, and multipolar mitoses. Red arrowheads indicate the corresponding mitotic aberrations indicated in the image.


A - C latency ( A ) , volume ( B ) , ( C ) of subcutaneous tumors generated by control Trf1 - downregulated K - RasG12V - transformed lung in athymic mice . D Representative of subcutaneous tumors .

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A-C The latency (A), volume (B), and weight (C) of subcutaneous tumors generated by control and Trf1-downregulated K-RasG12V-transformed lung cells in athymic mice.D Representative images of the subcutaneous tumors.


E Trf1 expression measured by qPCR in injected in generated subcutaneous tumors .

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E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.


F - H Number of Ki67 - positive ( F ) , number of γH2AX - positive ( G ) , number of active caspase - 3 - positive ( H ) per field in subcutaneous tumors .

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F-H Number of Ki67-positive (F), number of γH2AX-positive (G), and number of active caspase-3-positive (H) cells per field in the subcutaneous tumors.


I Representative of aberrant giant nuclei anaphase bridges in Trf1 - downregulated subcutaneous tumors compared to normal nuclei of control tumors .

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I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.


J TRF1 immunofluorescence shows downregulation of Trf1 in lung tumors of mice intravenously injected with control Trf1 - downregulated .

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J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.


K measured in lungs of mice intravenously injected with control Trf1 - downregulated . L Representative of lungs colonized by control Trf1 - downregulated , respectively .

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K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.


M - O Number of Ki67 - positive ( M ) , number of γH2AX - positive ( N ) , number of active caspase - 3 - positive ( O ) per field in lung tumors .

M-O Number of Ki67-positive (M), number of γH2AX-positive (N), and number of active caspase-3-positive (O) cells per field in the lung tumors.


P Trf1 expression measured by qPCR in A549 infected either with sh - scrambled or sh - Trf1 .

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P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.


Trf1 expression in indicated tissues of wild - type Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 .

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Trf1 expression levels in the indicated tissues of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Representative of TRF1 immunofluorescence of of TRF1 - positive in skin intestine sections of wild - type Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 .

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Representative images of TRF1 immunofluorescence and quantification of the percentage of TRF1-positive cells in skin and intestine sections of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Survival curve of wild - type Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 .

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Survival curve of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


of histological alterations observed in tamoxifen - treated Trf1lox / lox hUBC - CreERT2 mice 4 months after tamoxifen retrieval compared to their wild - type counterparts .

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Quantification of the histological alterations observed in tamoxifen-treated Trf1lox/lox hUBC-CreERT2 mice and 4 months after tamoxifen retrieval compared to their wild-type counterparts.


of blood populations in wild - type Trf1lox / lox hUBC - CreERT2mice subjected to a tamoxifen - containing diet for 7 after 3 4 months of tamoxifen retrieval .

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Quantification of blood cell populations in wild-type and Trf1lox/lox hUBC-CreERT2mice subjected to a tamoxifen-containing diet for 7 weeks and after 3 weeks and 4 months of tamoxifen retrieval.


Trf1 expression in blood , intestine , skin , bone marrow of Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet either for 7 or for 6 months after 3 tamoxifen retrieval compared to wild - type mice .

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Trf1 expression levels in blood, intestine, skin, and bone marrow of Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet either for 7 weeks or for 6 months and after 3 weeks tamoxifen retrieval compared to wild-type mice.


of TRF1 by immunofluorescence in lung - derived treated with DMSO , with 10 μM ETP - 47228 ( 24 h ) , with 10 μM ETP - 47037 ( 48 h ) . Representative are shown to right .

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Quantification of TRF1 levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with 10 μM ETP-47228 (24 h), and with 10 μM ETP-47037 (48 h). Representative images are shown to the right.


of γH2AX by immunofluorescence in lung - derived treated with DMSO , with ETP - 47228 ( 24 h ) , with ETP - 47037 ( 48 h ) . Representative are shown to right .

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Quantification of γH2AX levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with ETP-47228 (24 h), and with ETP-47037 (48 h). Representative images are shown to the right.


of telomere - induced foci ( TIFs ) by double immunofluorescence with anti - RAP1 anti - γH2AX antibodies . Representative are shown to right . arrowheads : colocalization of γH2AX RAP1 .

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Quantification of telomere-induced foci (TIFs) by double immunofluorescence with anti-RAP1 and anti-γH2AX antibodies. Representative images are shown to the right. White arrowheads: colocalization of γH2AX and RAP1.


Effect of different ETP - 47228 ETP - 47037 concentrations during 24 h on proliferation in lung - derived relative to growth of DMSO - treated .

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Effect of different ETP-47228 and ETP-47037 concentrations during 24 h on proliferation in lung tumor-derived cell line relative to the growth of DMSO-treated cells.


growth in allograft model ETP - 47037 or with ETP - 47228 .

Tumor growth quantification in allograft model ETP-47037 or with ETP-47228.


of growth relative to initial . Representative CT are shown to right . arrowhead indicates a within a highly inflammatory region .

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Quantification of tumor growth relative to initial tumor size. Representative CT images are shown to the right. The white arrowhead indicates a tumor within a highly inflammatory region.


of TRF1 by immunofluorescence in intestine lung tumors samples of mice treated with vehicle or with ETP - 47037 for 10 days . Representative are shown to right ( n = 4 ) .

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Quantification of TRF1 levels by immunofluorescence in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).


Number of showing γH2AX foci in intestine lung tumors samples of mice treated with vehicle or with ETP - 47037 for 10 days . Representative are shown to right ( n = 4 ) .

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Number of cells showing γH2AX foci in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).


A - C of number of ( A ) Ki67 - , ( B ) pan - p - H3 pattern - , ( C ) foci p - H3 pattern - positive in untreated ETP - 470037 - treated lungcarcinomas . data represent mean values obtained for three mice in each group . Error bars represent standard errors . D Representative Ki67 p - H3 .

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A-C Quantification of the number of (A) Ki67-, (B) pan-nuclear p-H3 pattern-, and (C) foci p-H3 pattern-positive cells in untreated and ETP-470037-treated lungcarcinomas. The data represent the mean values obtained for three mice in each group. Error bars represent standard errors.D Representative Ki67 and p-H3 images.


E Representative H & amp ; amp ; E of intestine samples corresponding to untreated ETP - 47037 - treated . High - magnification are shown to right indicating presence of normal mitosis , giant multinucleated aberrant mitotic figures .

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E Representative H&amp;E images of intestine samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown to the right indicating the presence of normal mitosis, giant multinucleated and aberrant mitotic figures.


F Representative H & amp ; amp ; E of bone marrow skin samples corresponding to untreated ETP - 47037 - treated . High - magnification are shown indicating presence of necrosis , hemosiderosis , multinucleated , giant nuclei . Bone marrow showed moderated aplasia .

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F Representative H&amp;E images of bone marrow and skin samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown indicating the presence of necrosis, hemosiderosis, multinucleated cells, and giant nuclei. Bone marrow showed moderated aplasia.


G Telomere in untreated ETP - 47037 - treated lung samples . Representative are shown to right .

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G Telomere length in untreated and ETP-47037-treated lung tumor samples. Representative images are shown to the right.


A Location of missense variants of PDIA1 ERp57 identified in ALS cases . PDI structure : catalytic a a ' domains containing active site motif CXXC ( purple green ) , non - catalytic domains b b ' containing ligand binding sites ( purple green ) , x - linker region ( gray ) . constructs used in this study contained a V5 - tag ( orange ) at C - terminus that was inserted previous to ER - retention signal ( gray ) .

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A Location of missense variants of PDIA1 and ERp57 identified in ALS cases. PDI primary structure: catalytic a and a' domains containing the active site motif CXXC sequence (dark purple and dark green), non-catalytic domains b and b' containing ligand binding sites (light purple and light green), and x-linker region (light gray). The constructs used in this study contained a V5-tag (orange) at the C-terminus that was inserted previous to the ER-retention signal (dark gray).


B Expression of PDI variants in zebrafish . Zebrafish at 1 - 2 stage were injected with sense mRNA coding for indicated PDIs ( PDIA1WT , PDIA1D292N PDIA1R300H : 200 pg / ; ERp57WT , ERp57D217N ERp57Q481K : 80 pg / ) . expression was confirmed by Western blot using anti - V5 antibody in total extracts at 24 h post - fertilization ( hpf ) . Actin was used as a loading control . In left panel 80 µg in right panel 120 µg extract were used .

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B Expression of PDI variants in zebrafish. Zebrafish embryos at 1-2 cell stage were injected with sense mRNA coding for the indicated PDIs (PDIA1WT, PDIA1D292N and PDIA1R300H: 200 pg/embryo; ERp57WT, ERp57D217N and ERp57Q481K: 80 pg/embryo). Protein expression was confirmed by Western blot analysis using anti-V5 antibody in total embryo extracts at 24 h post-fertilization (hpf). Actin was used as a loading control. In the left panel 80 µg protein and in the right panel 120 µg protein extract were used.


C D , Motoneuron defects induced in zebrafish after expression of indicated ALS - linked PDI mutants wild - type controls ( PDIA1WT PDIA1R300H : 80 pg mRNA / ; ERp57WT , ERp57D217N , ERp57D217N ERp57Q481K : 30 pgtba mRNA / ) . most frequent global induced by PDI injection are shown in lateral views of at 24 hpf ( left column ) . Black arrows indicate presence of curly tail / or shorter axis ( see details in Appendix Table S1 ) . Axon motoneuron was visualized using confocal microscopy in lateral views of trunk region in transgenic Tg ( Huc : Kaede ) zebrafish at 36 hpf ( middle right columns ) . in right column correspond to magnification views of rectangular regions depicted in left middle columns . Asterisks arrows point to examples of increased axonal branching reduced axonal , respectively .

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C and D, Motoneuron defects induced in zebrafish embryos after expression of the indicated ALS-linked PDI mutants and wild-type controls (PDIA1WT and PDIA1R300H: 80 pg mRNA/embryo; ERp57WT, ERp57D217N, ERp57D217N and ERp57Q481K: 30 pgtba mRNA/embryo). The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf (left column). Black arrows indicate the presence of curly tail and/or shorter axis phenotypes (see details in Appendix Table S1). Axon motoneuron morphology was visualized using confocal microscopy in lateral views of the trunk region in transgenic Tg(Huc:Kaede) zebrafish at 36 hpf (middle and right columns). Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns. Asterisks and arrows point to examples of increased axonal branching and reduced axonal length, respectively.


C D , Motoneuron defects induced in zebrafishembryos after expression of indicated ALS - linked PDI mutants wild - type controls ( PDIA1WT PDIA1R300H : 80 pg mRNA / ; ERp57WT , ERp57D217N , ERp57D217N ERp57Q481K : 30 pgtba mRNA / ) . most frequent global induced by PDI injection are shown in lateral views of at 24 hpf ( left column ) . Black arrows indicate presence of curly tail / or shorter axis ( see details in Appendix Table S1 ) . Axonmotoneuronmorphology was visualized using confocalmicroscopy in lateral views of trunk region in transgenic Tg ( Huc : Kaede ) zebrafish at 36 hpf ( middle right columns ) . in right column correspond to magnification views of rectangular regions depicted in left middle columns . Asterisks arrows point to examples of increased axonal branching reduced axonal , respectively .

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C and D, Motoneuron defects induced in zebrafishembryos after expression of the indicated ALS-linked PDI mutants and wild-type controls (PDIA1WT and PDIA1R300H: 80 pg mRNA/embryo; ERp57WT, ERp57D217N, ERp57D217N and ERp57Q481K: 30 pgtba mRNA/embryo). The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf (left column). Black arrows indicate the presence of curly tail and/or shorter axis phenotypes (see details in Appendix Table S1). Axonmotoneuronmorphology was visualized using confocalmicroscopy in lateral views of the trunk region in transgenic Tg(Huc:Kaede) zebrafish at 36 hpf (middle and right columns). Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns. Asterisks and arrows point to examples of increased axonal branching and reduced axonal length, respectively.


E F of motoneuron axon axon branching in 36hpf Tg ( Huc : Kaede ) injected with indicated PDIA1 ( D ) ERp57 ( E ) mutants .

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E and F Quantification of motoneuron axon length and axon branching in 36hpf Tg(Huc:Kaede) embryos injected with the indicated PDIA1 (D) and ERp57 (E) mutants.


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G Touch - evoked escape responses of 48 hpf zebrafish injected with ALS - linked PDI mutants ( Kabashi et al , 2011 ; Kabashi et al , 2009 ) . number of touches necessary to evoke an escape response ( top ) speed ( bottom , in mm / s ) of escape response was determined for each condition . total number of analyzed is indicated in parenthesis . Experiments of D - F were performed in selected sharing normal overall viability , to avoid unspecific effects of axial shortening or curvature in motoneuron / or behavior . Statistical analyses were performed using one - way ANOVA Bonferroni ´ s post hoc . Mean ± with only statistically significant p values are shown : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . Abbreviations : A ( anterior ) , D ( dorsal ) , P ( posterior ) , V ( ventral ) . Scale bars represent 500 m ( C D : a , d . g , j ) , 200 m ( C D : b , e , h , k ) , 100 m ( C D : c , f , i , l ) , 25 µm ( G - H ) .

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G Touch-evoked escape responses of 48 hpf zebrafish embryos injected with ALS-linked PDI mutants (Kabashi et al, 2011; Kabashi et al, 2009). The number of touches necessary to evoke an escape response (top) and speed (bottom, in mm/s) of the escape response was determined for each condition. The total number of analyzed embryos is indicated in parenthesis. Experiments of D-F were performed in selected animals sharing normal overall embryo morphology and viability, to avoid unspecific effects of axial shortening or curvature in motoneuron morphology and/or animal behavior. Statistical analyses were performed using one-way ANOVA and Bonferroni´s post hoc tests. Mean ± SEM with only statistically significant p values are shown: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Abbreviations: A (anterior), D (dorsal), P (posterior), V (ventral). Scale bars represent 500 m (C and D: a,d.g,j), 200 m (C and D: b,e,h,k), 100 m (C and D: c,f,i,l), and 25 µm (G-H).


A NSC34cells were transiently transfected with expression vectors for V5 - tagged wild - type mutant PDIs . After 48 h , overexpressed PDI variants was assessed under reducing conditions in an 8 % SDS - PAGE . An antibody detecting total PDIA1 ( endogenous mousePDIA1 exogenous humanV5 - tagged PDIA1 ) ( first panel ) . A second antibody detects only humanPDIA1 , therefore only V5 - tagged PDIA1 appears ( second panel ) . A mouse human specific antibody was used to detect total ERp57 ( third panel ) . Anti - V5 was used to detect exogenous PDI variants . Anti - β - actin was used as a loading control .

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A NSC34cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIs. After 48 h, overexpressed PDI variants was assessed under reducing conditions in an 8% SDS-PAGE. An antibody detecting total PDIA1 (endogenous mousePDIA1 and exogenous humanV5-tagged PDIA1) (first panel). A second antibody detects only humanPDIA1, therefore only V5-tagged PDIA1 appears (second panel). A mouse and human specific antibody was used to detect total ERp57 (third panel). Anti-V5 was used to detect the exogenous PDI variants. Anti-β-actin was used as a loading control.


B C NSC34 were transiently transfected with indicated PDIs constructs together with a GFP expression plasmid . were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce differentiation . Increased neurite outgrowth is indicated with arrow heads . B of average neurite lengths was performed , from three independent experiments were compiled . A minimum of 100 per experiment was analyzed . In addition , C of with neurites was quantified in three independent experiments ( right panel ) .

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B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid. Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. Increased neurite outgrowth is indicated with white arrow heads. B Quantification of the average primary neurite lengths was performed, all cells from three independent experiments were compiled. A minimum of 100 cells per experiment was analyzed. In addition, C the percentage of cells with neurites was quantified in the three independent experiments (right panel).


D rat ventral spinal cordneurons were prepared after 4 daysin vitro ( DIV ) transfected with GFP alone or together with indicated PDIs constructs . were fixed at 10 DIV SMI32 was performed to identify motoneurons . were taken total outgrowth of GFP SMI32 positive was quantified . Results are compiled from 3 independent experiments .

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D Primary rat ventral spinal cordneurons were prepared and after 4 daysin vitro (DIV) transfected with GFP alone or together with the indicated PDIs constructs. Cells were fixed at 10 DIV and SMI32 staining was performed to identify motoneurons. Images were taken and the total outgrowth of GFP and SMI32 positive cells was quantified. Results are compiled from 3 independent experiments.


E Human motoneurons were differentiated from human embryonic ( ESC ) HuESC 3 Hb9 : : GFP . Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDIs - expressing plasmids . Transduced were cultured for another 10 days . To identify motoneurons after lentiviral transduction , immunocytochemistry analyses were performed . of neurite outgrowth in different experimental conditions of GFP HB9 positive neurons were obtained . Neurite number assessment of of each neurite was performed in a similar manner as for rat motoneurons ( see methods ) . Four independent experiments were performed .

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E Human motoneurons were differentiated from the human embryonic stem cell (ESC) HuESC 3 Hb9::GFP line. Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDIs-expressing plasmids. Transduced cells were cultured for another 10 days. To identify motoneurons after lentiviral transduction, immunocytochemistry analyses were performed. Quantification of neurite outgrowth in the different experimental conditions of GFP and HB9 positive neurons were obtained. Neurite number and assessment of the length of each neurite was performed in a similar manner as for primary rat motoneurons (see methods). Four independent experiments were performed.


F NSC34 lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce differentiation . of with neurites was quantified in three independent experiments . A minimum of 100 per experiment was analyzed . Statistical analyses were performed using one - way ANOVA Bonferroni ´ s post hoc . Mean ± with only statistically significant p values are shown : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 .

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F NSC34 cell lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. The percentage of cells with neurites was quantified in the three independent experiments. A minimum of 100 cells per experiment was analyzed.Statistical analyses were performed using one-way ANOVA and Bonferroni´s post hoc tests. Mean ± SEM with only statistically significant p values are shown: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.


A PDI mutants form abnormal disulfide - dependent complexes . NSC34 were transiently transfected with expression vectors for V5 - tagged wild - type mutant PDIA1 . After 48 h , differential disulfide - dependent interactions / aggregations of overexpressed PDI variants was assessed under reducing ( + DTT ) non - reducing ( - DTT ) conditions in an 8 % SDS - PAGE . Anti - V5 was used for detection in Western blot .

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A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIA1. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.


B of PDIA1 structure to model effects of R300H mutation . close association between Arg300 located to b ' domain of PDIA1 with Trp396 located to a ' domain adjacent to active site motif CGHC ( designated as AS in yellow ) is shown in comparison to mutated version of PDIA1R300H highlighting same residues . A potential stabilization of interaction between b ' a ' domain is shown that may be caused by interaction of imidazole rings of mutated His300 with Trp396 .

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B Analysis of the PDIA1 structure to model the effects of the R300H mutation. The close association between Arg300 located to the b' domain of PDIA1 with Trp396 located to the a' domain adjacent to the active site motif CGHC (designated as AS in yellow) is shown in comparison to the mutated version of PDIA1R300H highlighting the same residues. A potential stabilization of the interaction between the b' and a' domain is shown that may be caused by the interaction of the imidazole rings of mutated His300 with Trp396.


C ALS - linked PDIA1 variants were generated as recombinant then analyzed by circular dichroism ( CD ) . Averages for CD spectroscopic scans of PDIA1WT mutants are shown .

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C ALS-linked PDIA1 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of PDIA1WT and mutants are shown.


D Averages for CD spectroscopic thermal denaturation of recombinant PDIA1WT mutants .

D Averages for CD spectroscopic thermal denaturation of recombinant PDIA1WT and mutants.


E Representative electrophoresis of Proteinase K - treated recombinant PDIA1 variants . Mass spectrometric of Proteinase K digested samples from total sample from in gel trypsin digested samples ( arrows indicate band 1 band 2 ) indicated differences in removal of x - linker region . Bottom panel : Ratios of band 1 to band 2 were quantified in 4 independent experiments .

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E Representative electrophoresis analysis of Proteinase K-treated recombinant PDIA1 variants. Mass spectrometric analysis of Proteinase K digested samples from total sample and from in gel trypsin digested protein samples (arrows indicate band 1 and band 2) indicated differences in the removal of the x-linker region. Bottom panel: Ratios of band 1 to band 2 were quantified in 4 independent experiments.


F Representative spectra of recombinant PDIA1b ' xWT fragments equivalent ALS - linked mutants ( n = 6 ) . Ratios from two peak areas change compared to PDIA1 b ' xWT . Both mutants analyzed show a significant shift in peak position compared to wild - type , but in opposite directions , suggesting that PDIA1D292N shifts equilibrium towards capped version ( x region over binding pocket ) PDIA1R300H mutant towards uncapped version ( Nguyen et al , 2008 ) .

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F Representative fluorescence spectra of recombinant PDIA1b'xWT fragments and the equivalent ALS-linked mutants (n=6). Ratios from the two peak areas and change compared to PDIA1 b'xWT. Both mutants analyzed show a significant shift in peak position compared to wild-type, but in opposite directions, suggesting that PDIA1D292N shifts equilibrium towards the capped version (x region over the binding pocket) and the PDIA1R300H mutant towards the uncapped version (Nguyen et al, 2008).


G activity of recombinant PDIA1 was measured in vitro using a BPTI refolding following by mass spectrometry . percentages of different BPTI species was calculated ( 6H , fully reduced ; 1S , one disulfide bond ; 2S , two disulfide bonds ; 3S , three disulfide bonds ) at time point 2 . 5 min in four independent experiments .

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G The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis. The percentages of different BPTI species was calculated (6H, fully reduced; 1S, one disulfide bond; 2S, two disulfide bonds; 3S, three disulfide bonds) at time point 2.5 min in four independent experiments.


H Measurement of H2O2 at ER lumen of living NSC34 . Left panel : Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen ( O2 ) , generating hydrogen peroxide ( H2O2 ) as a product of PDIs activity . Right panel : NSC34 were transiently co - transfected with ER luminal HyPer sensor indicated PDIs . After 48 h 490 / 420 nm was recorded for 2 min under basal conditions . Means derived from per condition ( n = 55 - 74 ) monitored in four independent experiments are shown .

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H Measurement of H2O2 levels at the ER lumen of living NSC34. Left panel: Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen (O2), generating hydrogen peroxide (H2O2) as a product of PDIs activity. Right panel: NSC34 cells were transiently co-transfected with ER luminal HyPer sensor and indicated PDIs. After 48 h the 490/420 nm fluorescence ratio was recorded for 2 min under basal conditions. Means and SEM derived from all cells per condition (n=55-74) monitored in four independent experiments are shown.


I HEK293T were transfected with expression vectors for V5 - tagged wild - type mutant PDIA1 , as well as empty vector . After 48 h , V5 - tagged were immunoprecipitated eluted with V5 peptide . interaction with endogenous ERO1Lα was analyzed by western blot . inputs elutions are shown as control . Right panel : of degree of interaction is presented .

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I HEK293T cells were transfected with expression vectors for V5-tagged wild-type and mutant PDIA1, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous ERO1Lα was analyzed by western blot. The inputs and elutions are shown as control. Right panel: Quantification of the degree of interaction is presented.


A PDI mutants form abnormal disulfide - dependent complexes . NSC34 were transiently transfected with expression vectors for V5 - tagged wild - type mutant ERp57 . After 48 h , differential disulfide - dependent interactions / aggregations of overexpressed PDI variants was assessed under reducing ( + DTT ) non - reducing ( - DTT ) conditions in an 8 % SDS - PAGE . Anti - V5 was used for detection in Western blot .

A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant ERp57. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.


B ALS - linked ERp57 variants were generated as recombinant then analyzed by circular dichroism ( CD ) . Averages for CD spectroscopic scans of recombinant ERp57WT mutants are shown .

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B ALS-linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of recombinant ERp57WT and mutants are shown.


C A thermal denaturation curve of ERp57WT mutants was performed .

C A thermal denaturation curve of ERp57WT and mutants was performed.


D Representative electrophoresis of Proteinase K treated ERp57 recombinant .

D Representative electrophoresis of Proteinase K treated ERp57 recombinant proteins.


E Surface Plasmon Resonance was performed to monitor affinity of recombinant ERp57 for recombinant CRT P - domain . KD values are expressed as of ERp57WT . Values are derived from four independent experiments . For absolute KD values please see Appendix Table S2 .

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E Surface Plasmon Resonance was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P-domain. KD values are expressed as percentage of ERp57WT. Values are derived from four independent experiments. For absolute KD values please see Appendix Table S2.


FHEK293T NSC34 were transfected with expression vectors for V5 - tagged wild - type mutant ERp57 , as well as empty vector . After 48 h , V5 - tagged were immunoprecipitated eluted with V5 peptide . interaction with endogenous calnexin ( CNX ) calreticulin ( CRT ) was analyzed by Western blot . inputs elutions are shown . Right panel : of degree of interaction is presented .

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FHEK293T NSC34 were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous calnexin (CNX) and calreticulin (CRT) was analyzed by Western blot. The inputs and elutions are shown. Right panel: Quantification of the degree of interaction is presented.


G gain of an N - glycosylation site of ERp57D217N was predicted after of since change of Asp217 to an Asn creates NXT / S consensus . Neuro2a were transfected with expression vectors for V5 - tagged wild - type mutant ERp57 , as well as empty vector treated with 1 µg / ml tunicamycin ( Tm ) for 20 h to inhibit N - glycosylation . Alternatively , extracts were digested with PNGase F possible removal of N - glycosylation was analyzed by Western blot using anti - V5 antibody .

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G The gain of an N-glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT/S consensus sequence. Neuro2a cells were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector and treated with 1 µg/ml tunicamycin (Tm) for 20 h to inhibit N-glycosylation. Alternatively, protein extracts were digested with PNGase F and the possible removal of the N-glycosylation was analyzed by Western blot using anti-V5 antibody.


A ERp57flox / flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient . of ERp57 in spinal cord were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) ERp57Nes - / - ( n = 4 ) mice . HSP90 were determined as a loading control . Right panel : of ERp57 was performed relative to Hsp90 .

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A ERp57flox/flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals. The levels of ERp57 protein in the spinal cord were monitored by Western blot. ERp57WT (n=4), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4) mice. HSP90 levels were determined as a loading control. Right panel: Quantification of ERp57 levels was performed relative to Hsp90 levels.


B measurements were performed for indicated time points in ERp57WT ( n = 50 ) , ERp57Nes + / - ( n = 32 ) ERp57Nes - / - ( n = 19 ) mice .

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B Body weight measurements were performed for indicated time points in ERp57WT (n=50), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=19) mice.


C Rotarod performance was performed ERp57WT ( n = 20 ) , ERp57Nes + / - ( n = 15 ) ERp57Nes - / - ( n = 8 ) mice .

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C Rotarod performance was performed ERp57WT (n=20), ERp57Nes+/- (n=15) and ERp57Nes-/- (n=8) mice.


D Hanging performance was assessed in ERp57WT ( n = 41 ) , ERp57Nes + / - ( n = 32 ) ERp57Nes - / - ( n = 12 ) mice .

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D Hanging test performance was assessed in ERp57WT (n=41), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=12) mice.


E Kaplan - Meier survival curve for ERp57Nes - / - mice ( N = 19 ) that prematurely died or had to be sacrificed because of health reasons between ages 22 73 days . Mean survival of this subgroup of was 57 days . ERp57WT ( n = 50 ) ERp57Nes + / - ( n = 32 ) mice are shown as a reference .

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E Kaplan-Meier survival curve for ERp57Nes-/- mice (N = 19) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days. Mean survival of this subgroup of animals was 57 days. ERp57WT (n=50) and ERp57Nes+/- (n=32) mice are shown as a reference.


F Histological of NeuN GFAP was performed in spinal cord tissue from ERp57WT ERp57Nes - / - mice in three per group using indirect immunofluorescence . nucleus was stained with Hoechst . Representative from one mouse per group are shown . Scale bar : 50 μm .

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F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes-/- mice in three animals per group using indirect immunofluorescence. The nucleus was stained with Hoechst. Representative images from one mouse per group are shown. Scale bar: 50 μm.


G Stereological of spinal cord from ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 4 ) ERp57Nes - / - ( n = 4 ) mice . Alternate series of sections from spinal cord of mice were either stained for Nissl ( top row ) or processed for immunohistochemistry for cholinergic marker Choline Acetyl Transferase ( ChAT , bottom row ) . nucleoli of motoneurons , as stained in Nissl series , were counted inside motoneurons pools previously defined using adjacent ChAT series ( contours not shown ) . densities of three genotypes are shown on right plots . No significant differences were found between genotypes . Scale bar represents 200 μm 50 μm on large inset , respectively .

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G Stereological analysis of the spinal cord from ERp57WT (n = 4), ERp57Nes+/- (n = 4) and ERp57Nes-/- (n = 4) mice. Alternate series of sections from the spinal cord of the mice were either stained for Nissl (top row images) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase (ChAT, bottom row images). The nucleoli of the motoneurons, as stained in the Nissl series, were counted inside the motoneurons pools previously defined using the adjacent ChAT series (contours not shown). Cell densities of the three genotypes are shown on the right plots. No significant differences were found between the genotypes. Scale bar represents 200 μm and 50 μm on large and inset images, respectively.


H Representative of spinal cord myelination of ERp57WT ERp57Nes - / - mice .

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H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes-/- mice.


A Representative EMG recordings of ERp57WT ( n = 16 ) , ERp57Nes - / + ( n = 12 ) ERp57Nes - / - ( n = 5 ) mice is presented . presence of positive sharp waves ( PSW ) in ERp57Nes + / - ERp57Nes - / - mice indicated muscle denervation .

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A Representative EMG recordings of ERp57WT (n=16), ERp57Nes-/+ (n=12) and ERp57Nes-/- (n=5) mice is presented. The presence of positive sharp waves (PSW) in ERp57Nes+/- and ERp57Nes-/- mice indicated muscle denervation.


B of NMJ muscle morphologies were performed from ERp57WT ( n = 5 ) , ERp57Nes + / - ( n = 5 ) ERp57Nes - / - ( n = 4 ) . Quantifications of endplate width , measuring most ventral region of diaphragms every 134 µm considering both sides of innervation profile . Twenty to forty measurements per diaphragm were obtained per .

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B Analysis of NMJ and muscle morphologies were performed from ERp57WT (n=5), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4). Quantifications of endplate width, measuring the most ventral region of the diaphragms every 134 µm considering both sides of the innervation profile. Twenty to forty measurements per diaphragm were obtained per animal.


C Whole - mounted diaphragms from ERp57WT ERp57Nes - / - mice were co - stained with anti - neurofilament ( red ) α - BTX to reveal postsynaptic densities ( green ) . 3D reconstructions ( lower panel ) of higher magnification . ERp57WT NMJs are fully innervated pretzel - like , whereas ERp57Nes - / - NMJs display less postsynaptic densities an incomplete or aberrantly distributed innervation pattern ( Movies EV4 - 7 ) .

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C Whole-mounted diaphragms from ERp57WT and ERp57Nes-/- mice were co-stained with anti-neurofilament (red) and α-BTX to reveal the postsynaptic densities (green). 3D reconstructions (lower panel) of higher magnification. ERp57WT NMJs are fully innervated pretzel-like, whereas ERp57Nes-/- NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern (Movies EV4-7).


D Representative 3D reconstructions of z - stacks showing five categories in which NMJs morphologies were grouped : Pretzel , fragmented , O shaped , C shaped , dismantled NMJs . Histogram of distribution frequency of > 200 different NMJ per is shown .

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D Representative 3D reconstructions of z-stacks showing the five categories in which NMJs morphologies were grouped: Pretzel, fragmented, O shaped, C shaped, and dismantled NMJs. Histogram of the distribution frequency of >200 different NMJ per animal is shown.


E Quantitative morphometry of > 60 NMJs in different genotypes analyzed .

E Quantitative morphometry of >60 NMJs in the different genotypes analyzed.


A of ERp57 in muscle were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) ERp57Nes - / - ( n = 4 ) mice . HSP90 was determined as loading control .

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A The levels of ERp57 protein in the muscle were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 was determined as loading control.


B Tibialis anterior from ERp57WT ( n = 5 ) , ERp57Nes + / - ( n = 5 ) , ERp57Nes - / - ( n = 4 ) mice were dissected cryosectioned ( 20 µm ) for of several parameters of muscle physiology . Upper panel : hyaluronic acid ( HA ) was performed to assess gross of muscle . Middle panel : Bismarck brown to monitor skeletal fibrosis . Lower panel : Cryosections were then double stained with wheat germ agglutinin ( WGA ) to visualize plasmamembrane nuclei . absence of central nuclei in genotypes suggests that ERp57 deficiency does not affect degeneration / regeneration cycles . are representative of each genotype where acquired in same anatomical region of TA .

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B Tibialis anterior muscles from ERp57WT (n = 5), ERp57Nes+/- (n = 5), and ERp57Nes-/- (n = 4) mice were dissected and cryosectioned (20 µm) for the analysis of several parameters of muscle physiology. Upper panel: hyaluronic acid (HA) staining was performed to assess the gross morphology of the muscle. Middle panel: Bismarck brown staining to monitor skeletal muscles fibrosis. Lower panel: Cryosections were then double stained with wheat germ agglutinin (WGA) to visualize plasmamembrane and nuclei. The absence of central nuclei in all genotypes suggests that ERp57 deficiency does not affect degeneration/regeneration cycles. Images are representative of each genotype and where acquired in the same anatomical region of the TA muscles.


C Tibialis anterior cryosections ( 20 µm ) from ERp57WT , ERp57Nes + / - , ERp57Nes - / - mice were stained for NADH - TR activity to identify oxidative myofibers ( upper panel ) . Representative of transversal cryosections show variable proportions of , intermediate positive in different genotypes : ( middle panel ) NADH - TR positive ( slow - twitch ) negative ( fast - twitch ) myofibers were quantified as of total for every genotype ( lower panel ) mean diameter of slow - fast - twitch muscle was evaluated at microscope using ImageJ software . Data represent average ± of > 300 per . Scale bars represent 50 m in B C .

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C Tibialis anterior cryosections (20 µm) from ERp57WT, ERp57Nes+/-, and ERp57Nes-/- mice were stained for NADH-TR activity to identify oxidative myofibers (upper panel). Representative micrographs of transversal cryosections show variable proportions of light, intermediate and dark positive staining in the different genotypes: (middle panel) NADH-TR positive (slow-twitch) and negative (fast-twitch) myofibers were quantified as percentage of total for every genotype (lower panel) The mean diameter of slow- and fast-twitch muscle fibers was evaluated at the light microscope using the ImageJ software. Data represent the average ± SEM of >300 fibers per animal.Scale bars represent 50 m in B and C.


A of synaptic SV2 , NMDAR2A , PSD95 Synaptophysin in cortex were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) ERp57Nes - / - ( n = 4 ) mice . HSP90 β - Actin were determined as loading controls .

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A The levels of synaptic proteins SV2, NMDAR2A, PSD95 and Synaptophysin in the cortex were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 and β-Actin were determined as loading controls.


B of SV2 monomer ( left panel ) SV2 high molecular species ( right panel ) . β - Actin was used as reference .

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B Quantification of SV2 monomer (left panel) and SV2 high molecular weight species (right panel). β-Actin was used as the reference.


( A ) volume at indicated time points after CT26 transplant in mice treated or not with LPS n ( number of per group ) = 6 .

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(A) Tumor volume at the indicated time points after CT26 cell transplant in mice treated or not with LPS n (number of animals per group)=6.


( B ) volume in mice depleted of DCs or NK before transplant LPS administration n ( number of per group ) = 5 .

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(B) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n (number of animals per group)=5.


( C ) volume in SCIDmice treated or not with LPS n ( number of per group ) = 5 .

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(C) Tumor volume in SCIDmice treated or not with LPS n (number of animals per group)=5.


( D ) volume at indicated time point after repetitive LPS injections as depicted in bottom scheme n ( number of per group ) = 6 .

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(D) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n (number of animals per group)=6.


( E ) of NK within in mice treated or not with LPS depleted or not of DCs n ( number of per group ) ≥ 4 . NT , control ; DT , diphtheria toxin treated to deplete DCs ; LPS , mice treated with LPS . Error bars depict . Statistical significance was determined with a two - tail t . NT , untreated .

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(E) Percent of NK cells within the tumor in mice treated or not with LPS and depleted or not of DCs n (number of animals per group) ≥ 4. NT, control; DT, diphtheria toxin treated animals to deplete DCs; LPS, mice treated with LPS. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


( A ) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in scheme at bottom of panel .

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(A) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel.


( B ) Detection of blood vessels by immunohistochemistry using anti - CD31 antibody ( brown ) in frozen sections of tumors from mice treated or not with with LPS as in scheme in ( A ) . Nuclei are stained with hematoxylin . Two representative sections are shown , magnification 20X . Left panel scale bars , 500m ; right panel scale bars 100 m . of vessel mean dimensions coverage by vessels is also shown . Three sections from three tumors have been analyzed . Error bars depict . Statistical significance was determined with a two - tail t . NT , untreated .

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(B) Detection of blood vessels by immunohistochemistry using anti-CD31 antibody (brown staining) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in (A). Nuclei are stained with hematoxylin. Two representative sections are shown, magnification 20X. Left panel scale bars, 500m; right panel scale bars 100 m. Quantification of vessel mean dimensions and area coverage by vessels is also shown. Three sections from three tumors have been analyzed. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


( A , B ) Absolute numbers of IFN - γ + total NK in draining lymph nodes at indicated time points before after LPS administration in mice treated or not with FTY720 ( 25g / mouse ) to reduce ingress of lymphocytes in efferent lymphatics .

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(A,B) Absolute numbers of IFN-γ+ and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 (25g/mouse) to reduce the ingress of lymphocytes in the efferent lymphatics.


( C ) volume in mice treated or not with LPS / or FTY720 according to scheme .

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(C) Tumor volume in mice treated or not with LPS and/or FTY720 according to the scheme.


( D ) of IFN - γ + NK within in mice treated or not with LPS with FTY720 . n ( number of per group ) = 4 . Error bars depict . FTY : FTY720 . Statistical significance was determined with a two - tail t . NT , untreated .

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(D) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with FTY720. n (number of animals per group)=4. Error bars depict SEM. FTY: FTY720. Statistical significance was determined with a two-tail t test. NT, untreated.


( A ) Absolute numbers of NK in one of 4 draining lymph nodes in mice treated or not with anti - CD62L antibody to inhibit NK cel ingress in lymph node from blood . Where indicated mice were challenged with LPS .

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(A) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti-CD62L antibody to inhibit NK cel ingress in the lymph node from blood. Where indicated mice were challenged with LPS.


( B ) Absolute numbers of IFN - γ + NK in one of 4 draining lymph nodes before after LPS administration in mice in which NK entry in lymph node was blocked or not by anti - CD62L treatment .

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(B) Absolute numbers of IFN-γ+ NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti-CD62L treatment.


( C ) ( left panel ) Absolute numbers of endogenous adoptively transferred ( CFSE + ) NK in draining lymph node 5 hours after LPS administration ; ( right panel ) of endogenous adoptively transferred NK on total lymphocytes in draining lymph node 5 hours after LPS administration .

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(C) (left panel) Absolute numbers of endogenous and adoptively transferred (CFSE+) NK cells in the draining and contralateral lymph node 5 hours after LPS administration; (right panel) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration.


( D ) Flow cytometry of LPS draining lymph node from mice that received CFSE labeled at moment of LPS administration . Lymph node were stained with anti - CD3 , anti - DX5 anti - IFN - γ antibodies performed on gated CD3 - DX5high . One representative of 4 independent experiments is shown .

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(D) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration. Lymph node cells were stained with anti-CD3, anti-DX5 and anti-IFN-γ antibodies and the analysis performed on gated CD3-DX5high cells. One representative of 4 independent experiments is shown.


( E ) volume in mice treated or not with LPS , where indicated mice received anti - CD62L antibody 12h before LPS treatment .

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(E) Tumor volume in mice treated or not with LPS, where indicated mice received anti-CD62L antibody 12h before LPS treatment.


( F ) of IFN - γ + NK within in mice treated or not with LPS with anti - CD62L antibody .

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(F) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with the anti-CD62L antibody.


( A ) Representative image of an NK ( red ) interacting with four DCs ( green ) . ( B , C , D ) Plot of confinement ( CR , black squares ) , normalized NK speed Vthr ( ti ) ( red circles ) digitalized NK - DC , DistD ( ti ) ( green triangles ) as a function of time for NK reported in image . B , C D panels correspond respectively to interaction with DC1 , DC2 DC4 displayed in ( A ) . For reference also normalized Dist ( ti ) is reported in plots ( blue down triangles ) . time periods in which three conditions discussed in text are fulfilled are marked with red striped rectangles in plots . green up - triangles indicate parameter TonToff defined as TonToff = - 1 if Dist ( ti ) < d = 25 µm TonToff = - 1 if Dist ( ti ) d indicate putative interactions according to a more simplified algorithm on DC - NK alone .

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(A) Representative image of an NK cell (red) interacting with four DCs (green). (B, C, D) Plot of the confinement ratio (CR, black squares), the normalized NK speed Vthr(ti) (red circles) and the digitalized NK-DC distance, DistD(ti) (green triangles) as a function of time for the NK cell reported in the image. The B, C and D panels correspond respectively to the interaction with the DC1, DC2 and DC4 displayed in (A). For reference also the normalized distance Dist(ti) is reported in the plots (blue down triangles). The time periods in which all the three conditions discussed in the text are fulfilled are marked with red striped rectangles in the plots. The green up-triangles indicate the parameter TonToff defined as TonToff = -1 if Dist(ti) < d = 25 µm and TonToff = -1 if Dist(ti) d and indicate the putative interactions according to a more simplified algorithm based on the DC-NK cell distance alone.


( A ) NK DC trajectories as they are tracked by Volocity software on 4D volumes collected in lymph nodes on TPE microscope . of shows interaction between an NK ( red ) a DC ( green ) . In top panels NK moves according to a directional random motion while in lower left middle panels NK seems to recognize DC to go back . In right lower panel stable contact DC - NK is clearly visible . ( B ) Time duration of DC - NK contacts in steady state ( - LPS ) inflammatory ( + LPS ) conditions . Bars indicate mean of distribution . of showing long interaction times ( ≥ 15 min ) are shown . ( C ) NK 3D velocities . 3D velocities of NK taking contacts with DCs before ( black ) 5 h ( green ) after LPS treatment .

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(A) NK and DC trajectories as they are tracked by Volocity software on the 4D volumes collected in lymph nodes on the TPE microscope. The sequence of images shows the interaction between an NK cell (red) and a DC (green). In the top panels the NK cell moves according to a directional random motion while in the lower left and middle panels the NK cell seems to recognize the DC and to go back. In the right lower panel the stable contact DC-NK cell is clearly visible. (B) Time duration of the DC-NK contacts in steady state (-LPS) and inflammatory (+LPS) conditions. Bars indicate the mean of the distribution. The percent of cells showing long interaction times (≥ 15 min) are shown. (C) NK cell 3D velocities. 3D velocities of NK cells taking contacts with DCs before (black) and 5 h (green) after LPS treatment.


( A ) Differential interference contrast ( left panels ) confocal microscopy of IL - 18 ( right panels ) in a DC / NK conjugate after 2 hours of coculture in presence of LPS in unstimulated ( NT ) . Representative from 3 experiments are shown , magnification 63X . Scale bars , 4 . 36 m .

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(A) Differential interference contrast images (left panels) and confocal microscopy analysis of IL-18 (right panels) in a DC/NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells (NT). Representative images from 3 experiments are shown, magnification 63X. Scale bars, 4.36 m.