F ) Epidermal thickness of WT Pvt1 KO skin grafts was quantified presented as box whisker plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) . n = 18 , P < 0 . 01 ( Student ' s t - ) .

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F) Epidermal thickness of WT and Pvt1 KO skin grafts was quantified and presented as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=18, P<0.01 (Student's t-test).


G ) Number of p63 - positive in WT cKO skin was quantified shown as bar graph . n = 5 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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G) Number of p63-positive cells in WT and cKO skin was quantified and shown as bar graph. n=5, P<0.01 (Student's t-test). Error bar represents S.D.


H ) microscopy demonstrates different survival capability of WT Pvt1 inducible KO with or without Doxycycline ( Dox ) treatment .

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H) Fluorescence microscopy demonstrates different survival capability of WT and Pvt1 inducible KO cells with or without Doxycycline (Dox) treatment.


I ) of WT Pvt1 inducible KO in co - culture model was quantified shown as dot plots . n = 8 , P < 0 . 01 ( Student ' s t - ) for KO with Dox treatment compared with WT or KO without Dox stimulation at both Day 7 14 . Error bar represents S . D .

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I) Ratio of WT and Pvt1 inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with Dox treatment compared with WT cells or KO cells without Dox stimulation at both Day 7 and 14. Error bar represents S.D.


A ) Interaction between Pvt1 MYC was determined by immunoprecipitation followed with RT - PCR . Note significant decrease in Pvt1 MYC interaction upon FTO treatment ( left panel ) , calcium shift - induced differentiation ( middle panel , Hi : high calcium ) , Pvt1 methylation site mutations ( right panel ) . n = 3 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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A) Interaction between Pvt1 and MYC was determined by immunoprecipitation followed with RT-PCR. Note significant decrease in Pvt1 and MYC interaction upon FTO treatment (left panel), calcium shift-induced differentiation (middle panel, Hi: high calcium), and Pvt1 methylation site mutations (right panel). n=3, P<0.01 (Student's t-test). Error bar represents S.D.


B ) Immunoblots show decreased MYC level in differentiated keratinocytes Mettl14 KO .

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B) Immunoblots show decreased MYC protein level in differentiated keratinocytes and Mettl14 KO cells.


Undifferentiated WT Mettl14 KO ( C ) were treated with cycloheximide . lysates were collected at 0 , 30 , 60 , 90 , 120 , 180 , 300 minutes post treatment subjected to immunoblotting with different antibodies as indicated .

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Undifferentiated WT and Mettl14 KO cells (C) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.


undifferentiated WT Pvt1 KO ( D ) were treated with cycloheximide . lysates were collected at 0 , 30 , 60 , 90 , 120 , 180 , 300 minutes post treatment subjected to immunoblotting with different antibodies as indicated .

undifferentiated WT and Pvt1 KO cells (D) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.


WT before after calcium shift - induced differentiation ( E ) were treated with cycloheximide . lysates were collected at 0 , 30 , 60 , 90 , 120 , 180 , 300 minutes post treatment subjected to immunoblotting with different antibodies as indicated .

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WT cells before and after calcium shift-induced differentiation (E) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.


( F ) Skin organoids derived from Mettl14 KO or KO rescued with exogenous expression of MYC were grafted to nude mice . regenerated skin was analyzed by H / E .

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(F) Skin organoids derived from Mettl14 KO cells or KO cells rescued with exogenous expression of MYC were grafted to nude mice. The regenerated skin was analyzed by H/E staining.


( G ) Epidermal thickness of KO KO skin rescued with MYC expression was quantified presented as box whisker plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) . n = 18 , P < 0 . 01 ( Student ' s t - ) .

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(G) Epidermal thickness of KO and KO skin rescued with MYC expression was quantified and presented as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=18, P<0.01 (Student's t-test).


( H ) Number of p63 - positive in KO KO skin rescued with MYC expression was quantified shown as bar graph . n = 5 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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(H) Number of p63-positive cells in KO and KO skin rescued with MYC expression was quantified and shown as bar graph. n=5, P<0.01 (Student's t-test). Error bar represents S.D.


( I ) microscopy demonstrates different survival capability of WT Mettl14 KO with inducible expression of MYC .

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(I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 KO cells with inducible expression of MYC.


( J ) of WT Mettl14 KO with inducible expression of MYC in co - culture model was quantified shown as dot plots . n = 8 , P < 0 . 01 ( Student ' s t - ) for KO without Dox treatment ( no MYC expression ) compared with WT or KO with Dox stimulation ( exogenous MYC expression ) at both Day 7 14 . Error bar represents S . D .

O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O

(J) Ratio of WT and Mettl14 KO cells with inducible expression of MYC in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells without Dox treatment (no MYC expression) compared with WT cells or KO cells with Dox stimulation (exogenous MYC expression) at both Day 7 and 14. Error bar represents S.D.


a , b , of HeLa stably expressing YFP fused to indicated galectins infected with S . Typhimurium for 1 h . a , of coated by indicated galectins . YFP - positive were counted by microscopy . Mean standard deviation ( s . d . ) of triplicate HeLa cultures , n > 100 per coverslip . b , Confocal microscopy . Arrowheads , shown in insets . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole .

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a, b, Analysis of HeLa cells stably expressing YFP fused to the indicated galectins and infected with S. Typhimurium for 1 h. a, Percentage of bacteria coated by the indicated galectins. YFP-positive bacteria were counted by microscopy. Mean and standard deviation (s.d.) of triplicate HeLa cultures, n > 100 bacteria per coverslip. b, Confocal microscopy. Arrowheads, bacteria shown in insets. DAPI, 4′,6-diamidino-2-phenylindole.


c , Kinetics of fold replication for S . Typhimurium in HeLa transfected with indicated siRNAs . were counted on basis of their ability to form colonies on agar plates . Mean s . d . of triplicate HeLa cultures duplicate colony . siRNAs are further characterized in Supplementary Fig . 2a - c . * * P 0 . 01 , Student ' s t - . Scale bar , 10 µm .

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c, Kinetics of fold replication for S. Typhimurium in HeLa cells transfected with the indicated siRNAs. Bacteria were counted on the basis of their ability to form colonies on agar plates. Mean and s.d. of triplicate HeLa cultures and duplicate colony counts. siRNAs are further characterized in Supplementary Fig. 2a-c. **P  0.01, Student's t-test. Scale bar, 10 µm.


a , LUMIER binding : normalized between luciferase activity bound to beads present in lysates . Lysates of 293ET expressing NDP52 , p62 or optineurin each fused to luciferase , indicated Flag - tagged galectins were incubated with anti - Flag beads . Flag - tagged are further characterized in Supplementary Fig .

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a, LUMIER binding assay: normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing NDP52, p62 or optineurin each fused to luciferase, and the indicated Flag-tagged galectins were incubated with anti-Flag beads. Flag-tagged proteins are further characterized in Supplementary Fig. 4a


b , Lysates of 293ET , expressing Flag - tagged as indicated , were immunoprecipitated with anti - Flag beads . Lysates immunoprecipitates ( IP ) were blotted for presence of Flag - tagged endogenous NDP52 .

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b, Lysates of 293ET cells, expressing Flag-tagged proteins as indicated, were immunoprecipitated with anti-Flag beads. Lysates and immunoprecipitates (IP) were blotted for the presence of Flag-tagged proteins and endogenous NDP52.


c , Confocal of HeLa infected with S . Typhimurium for 1 h stained with antisera against NDP52 galectin 8 . Arrowheads , shown in insets .

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c, Confocal images of HeLa cells infected with S. Typhimurium for 1 h and stained with antisera against NDP52 and galectin 8. Arrowheads, bacteria shown in insets.


d , Co - localization of NDP52 with galectin - positive in HeLa stably expressing YFP fused to indicated galectins , infected with S . Typhimurium stained with NDP52 antiserum 1 h after infection . Mean s . d . of duplicate HeLa cultures , n > 100 per coverslip , representative of two independent experiments . Scale bar , 10 µm .

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d, Co-localization of NDP52 with galectin-positive bacteria in HeLa cells stably expressing YFP fused to the indicated galectins, infected with S. Typhimurium and stained with NDP52 antiserum 1 h after infection. Mean and s.d. of duplicate HeLa cultures, n > 100 bacteria per coverslip, representative of two independent experiments. Scale bar, 10 µm.


a , of S . Typhimurium coated by indicated galectins . HeLa stably expressing YFP - tagged galectins were treated with indicated siRNAs . YFP - positive were counted by microscopy at 1 h p . i . siRNAs are further characterized in Supplementary Fig . 2 .

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a, Percentage of S. Typhimurium coated by the indicated galectins. HeLa cells stably expressing YFP-tagged galectins were treated with the indicated siRNAs. YFP-positive bacteria were counted by microscopy at 1 h p.i. siRNAs are further characterized in Supplementary Fig. 2.


b , of HeLa treated with indicated siRNAs stained with NDP52 antiserum . NDP52 - positive were counted by microscopy 1 h after infection with S . Typhimurium . siRNAs are further characterized in Supplementary Fig . 2 .

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b, Analysis of HeLa cells treated with the indicated siRNAs and stained with NDP52 antiserum. NDP52-positive bacteria were counted by microscopy 1 h after infection with S. Typhimurium. siRNAs are further characterized in Supplementary Fig. 2.


c , of coated by indicated galectin 8 alleles . HeLa stably expressing indicated galectin 8 alleles fused to YFP were infected with S . Typhimurium . YFP - positive S . Typhimurium were counted by microscopy at 75 min p . i . WT , wild type .

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c, Percentage of bacteria coated by the indicated galectin 8 alleles. HeLa cells stably expressing the indicated galectin 8 alleles fused to YFP were infected with S. Typhimurium. YFP-positive S. Typhimurium were counted by microscopy at 75 min p.i. WT, wild type.


d , Binding of galectin 8 to HeLa . indicated HeLa were incubated with His - GST - ubiquitin ( Ub ) , His - GST - galectin 8 or buffer as indicated , followed by murine anti - His antibody PE - labelled anti - mouse serum .

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d, Binding of galectin 8 to bacteria and HeLa cells. The indicated bacteria and HeLa cells were incubated with His-GST-ubiquitin (Ub), His-GST-galectin 8 or buffer as indicated, followed by murine anti-His antibody and PE-labelled anti-mouse serum.


e , of S . Typhimurium coated by indicated galectins . Wild - type CHO mutant Lec3 . 2 . 8 . 1 stably expressing YFP - tagged galectins were infected with S . Typhimurium . YFP - positive were counted by microscopy at 1 h p . i .

O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O O O O

e, Percentage of S. Typhimurium coated by the indicated galectins. Wild-type CHO cells and mutant Lec3.2.8.1 cells stably expressing YFP-tagged galectins were infected with S. Typhimurium. YFP-positive bacteria were counted by microscopy at 1 h p.i.


f , Confocal of HeLa expressing indicated YFP - tagged galectins . were left untreated or were exposed to hypertonic conditions , with or without ( w / o ) PEG as indicated , followed by hypotonic shock .

O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

f, Confocal images of HeLa cells expressing the indicated YFP-tagged galectins. Cells were left untreated or were exposed to hypertonic conditions, with or without (w/o) PEG as indicated, followed by hypotonic shock.


g , Fold replication of S . Typhimurium in HeLa expressing indicated galectin 3 variants transfected with indicated siRNAs . At 2 h 6 h after infection , were lysed counted on basis of their ability to form colonies on agar plates . Galectin 3 are further characterized in Supplementary Fig . 4b . Mean s . d . of duplicate coverslips > 100 counted per coverslip . Data are representative of at least two repeats . * P < 0 . 05 , Student ' s t - . Scale bar , 10 µm .

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g, Fold replication of S. Typhimurium in HeLa cells expressing the indicated galectin 3 variants and transfected with the indicated siRNAs. At 2 h and 6 h after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Galectin 3 proteins are further characterized in Supplementary Fig. 4b. Mean and s.d. of duplicate coverslips>100 bacteria counted per coverslip. Data are representative of at least two repeats. *P< 0.05, Student's t-test. Scale bar, 10 µm.


a , Confocal micrograph of HeLa expressing GFP - LC3 mCherry - galectin - 8 , stained for NDP52 1 h after infection with S . Typhimurium . lower right panel contains a scan along yellow in merge inset . Arrowheads , shown in insets .

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a, Confocal micrograph of HeLa cells expressing GFP-LC3 and mCherry-galectin-8, stained for NDP52 1 h after infection with S. Typhimurium. The lower right panel contains a fluorescence line scan along the yellow line in the merge inset. Arrowheads, bacteria shown in insets.


b , of GFP - LC3 - positive S . Typhimurium at 1 h p . i . in HeLa treated with indicated siRNAs .

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b, Percentage of GFP-LC3-positive S. Typhimurium at 1 h p.i. in HeLa cells treated with the indicated siRNAs.


c , of positive for NDP52 . HeLa expressing indicated NDP52 variants fused to YFP were infected with S . Typhimurium .

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c, Percentage of bacteria positive for NDP52. HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S. Typhimurium.


d , of positive for indicated markers . HeLa , either wild type or expressing YFP - galectin - 8 as indicated , were infected with S . Typhimurium . Ubiquitin was detected by antibody .

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d, Percentage of bacteria positive for the indicated markers. HeLa cells, either wild type or expressing YFP-galectin-8 as indicated, were infected with S. Typhimurium. Ubiquitin was detected by antibody staining.


e , HeLa treated with indicated siRNAs were infected with S . Typhimurium stained for NDP52 at indicated time points . were counted by microscopy at indicated time points . Graphs , representing at least two independent repeats , show mean s . d . of duplicate coverslips for which > 200 were counted . * P 0 . 05 , Student ' s t - . Scale bar , 10 µm .

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e, HeLa cells treated with the indicated siRNAs were infected with S. Typhimurium and stained for NDP52 at the indicated time points. Fluorescent bacteria were counted by microscopy at the indicated time points. Graphs, representing at least two independent repeats, show mean and s.d. of duplicate coverslips for which >200 bacteria were counted. *P  0.05, Student's t-test. Scale bar, 10 µm.


( A ) Representative transmission electron microscope ( ) image showing presence of EVs in CSF in two independent experiments .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-TISSUE O O O O O

(A) Representative transmission electron microscope (TEM) image showing the presence of EVs in the CSF in two independent experiments.


( B ) NanoSightquantification of amount of particles in CSF 0 , 1 , 2 , 4 6 hours after i . p . LPS injection ( n = 3 - 5 ) .

O O O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(B) NanoSightquantification of the amount of particles in the CSF 0, 1, 2, 4 and 6 hours after i.p. LPS injection (n=3-5).


( C ) distribution of EVsin vivo in CSF before ( black ; n = 5 ) 6 h after ( grey ; n = 3 ) LPS treatment determined by NanoSight .

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(C) Size distribution of the EVsin vivo in the CSF before (black; n=5) and 6 h after (grey; n=3) LPS treatment determined by NanoSight analysis.


( D - G ) Quantitative real - time polymerase chain reaction of miR - 1a ( D ) , miR - 9 ( E ) , miR - 146a ( F ) , miR - 155 ( G ) ( n = 4 ) . was isolated from pooled CSF ( 50 µl ) from different mice ( n = 3 ) . 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 .

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(D-G) Quantitative real-time polymerase chain reaction analysis of miR-1a (D), miR-9 (E), miR-146a (F), and miR-155 (G) (n=4). RNA was isolated from pooled CSF (50 µl) from different mice (n=3). 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.


( A - B ) In vitro ( A ) distribution ( B ) of EVs isolated from conditioned medium of CPE grown in transwell system after 12 h in absence ( black ) or presence ( grey ) of LPS ( n = 5 ) determined by NanoSight .

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(A-B) In vitro quantification (A) and size distribution (B) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence (black) or presence (grey) of LPS (n=5) determined by NanoSight analysis.


( C - E ) TaqMan qPCR for of miR - 9 ( C ) , miR - 146a ( D ) miR - 155 ( E ) on exosomal pellet isolated from conditioned medium of CPE grown in a transwell system stimulated for 12 h with LPS ( n = 3 ) .

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(C-E) TaqMan qPCR assay for the quantification of miR-9 (C), miR-146a (D) and miR-155 (E) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS (n=3).


( F - I ) of miRNAs miR - 1a ( F ) , miR - 9 ( G ) , miR - 146a ( H ) miR - 155 ( I ) by TaqMan qPCR from CPE grown in a transwell system without or with LPS stimulation ( n = 3 ) . Significance are indicated on graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-I) Quantification of the miRNAs miR-1a (F), miR-9 (G), miR-146a (H) and miR-155 (I) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation (n=3). Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A ) In vitro of EVs isolated from conditioned medium of LPS - stimulated CPE grown in a transwell system in absence or presence of exosome inhibitor GW4869 ( n = 3 ) .

O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O

(A) In vitro quantification of EVs isolated from conditioned medium of LPS-stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 (n=3).


( B - D ) TaqMan of miRNAs miR - 9 ( B ) , miR - 146a ( C ) miR - 155 ( D ) in supernatant of LPS - stimulated CPE grown in a transwell system either left untreated or pretreated with GW4869 to inhibit exosome secretion ( n = 3 ) . miR - 1a were below detection limit .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

(B-D) TaqMan assay quantification of the miRNAs miR-9 (B), miR-146a (C) and miR-155 (D) in supernatant of LPS-stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion (n=3). miR-1a levels were below detection limit.


( E - H ) TaqMan of miRNAs miR - 1a ( E ) , miR - 9 ( F ) , miR - 146a ( G ) miR - 155 ( H ) in lysate of LPS - stimulated CPE grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion ( n = 3 ) . 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 .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-H) TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G) and miR-155 (H) in cell lysate of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n=3). 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.


( A ) Representative confocal of CD63 , RAB5 ANXA2 ( red ) in choroid plexus ( CP ) 0 , 4 8 h after LPS treatment . Hoechst ( blue ) was used to stain nucleus . dotted indicates ependymal that ventricle square boxes indicate zoomed insert displayed at right corner of each image . Scalebar = 100 µm .

O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O O O O

(A) Representative confocal images of CD63, RAB5 and ANXA2 (red) in the choroid plexus (CP) 0, 4 and 8 h after LPS treatment. Hoechst (blue) was used to stain the nucleus. The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image. Scalebar = 100 µm.


( B - C ) Representative showing presence of MVBs in CPE before ( B ) 6 hours after ( C ) LPS administration in vivo . arrow heads point to exosomes present in MVBs ( n = 3 ) . Scale bar = 9 µm .

O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O

(B-C) Representative TEM images showing the presence of MVBs in the CPE cells before (B) and 6 hours after (C) LPS administration in vivo. White arrow heads point to exosomes present in MVBs (n=3). Scale bar = 9 µm.


( D - F ) of amount of MVBs per section ( D ) , amount of exosomes per MVB ( E ) , amount of exosomes per section ( F ) , on .

O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O

(D-F) Quantification of amount of MVBs per cell section (D), amount of exosomes per MVB (E), and amount of exosomes per cell section (F), based on TEM analysis.


( 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 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) .

O O O B-EXP_ASSAY O B-TISSUE O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O

(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 ) .

O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O

(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 .

O O O B-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O O O

(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 .

O O O B-EXP_ASSAY O B-SUBCELLULAR O B-TISSUE O O O O O O O O O O O O O O O B-ORGANISM O O O O O

(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 .

O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O

(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 .

O O O B-EXP_ASSAY O B-SUBCELLULAR O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O

(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 .

O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O B-ORGANISM O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-ORGANISM O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-CELL_TYPE O O O B-GENEPROD O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O

(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 .

O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-GENEPROD O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-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 ) .

O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(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 ) .

O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(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 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O B-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O

(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 ) .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-ORGANISM O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O

(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 .

O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-TISSUE O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O

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 .

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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 .

O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

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 .

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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 .

O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E 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 ) .

O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

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).