F T98G were challenged or not with dsDNA for 6 h , in presence or not of NU7441 - PKcs inhibitor , prior WB using indicated antibodies .

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F T98G cells were challenged or not with dsDNA for 6 h, in the presence or not of the NU7441 DNA-PKcs inhibitor, prior WB analysis using indicated antibodies.


I T98G were treated or not with 0 . 16 µM camptothecin ( CPT ) for 48 h prior to immunofluorescence using dsDNA - 53BP1 - specific antibodies , DAPI ( n = 3 independent experiments ) .

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I T98G cells were treated or not with 0.16 µM camptothecin (CPT) for 48 h prior to immunofluorescence analysis using dsDNA- and 53BP1-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).


J Whole extracts from T98G treated or not for 72 h with 0 . 16 µM CPT were analyzed by WB using indicated antibodies .

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J Whole cell extracts from T98G cells treated or not for 72 h with 0.16 µM CPT were analyzed by WB using indicated antibodies.


A T98G were treated or not with dsDNA for 6 h prior to IF using - PKcs - pDNA - PKcs - specific antibodies , DAPI ( n = 3 independent experiments ) .

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A T98G cells were treated or not with dsDNA for 6 h prior to IF analysis using DNA-PKcs- and pDNA-PKcs-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).


B Whole extracts from T98G were incubated with 80nt - long biotinylated ssDNA or dsDNA prior to pull - down using streptavidin - affinity beads . Input eluates were analyzed by WB using indicated antibodies .

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B Whole cell extracts from T98G cells were incubated with 80nt-long biotinylated ssDNA or dsDNA prior to pull-down using streptavidin-affinity beads. Input and eluates were analyzed by WB using indicated antibodies.


C T98G were treated with non - targeting ( CTRL ) or KU70 - targeting siRNAs prior to whole extract preparation , pull - down as in B . Inputs eluates were analyzed by WB using indicated antibodies .

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C T98G cells were treated with non-targeting (CTRL) or KU70-targeting siRNAs prior to whole cell extract preparation, and pull-down as in B. Inputs and eluates were analyzed by WB using indicated antibodies.


A T98G stably expressing cGAS ( T98GcGAS ) or not ( T98GEmpty ) were transfected or not with dsDNA for 6 h prior to whole extraction WB using indicated antibodies .

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A T98G cells stably expressing cGAS (T98GcGAS) or not (T98GEmpty) were transfected or not with dsDNA for 6 h prior to whole cell extraction and WB analysis using indicated antibodies.


C T98GEmpty T98GcGAS were transfected or not with biotinylated dsDNA prior to whole extraction pull - down using streptavidin - affinity beads . Inputs eluates were analyzed by WB using indicated antibodies .

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C T98GEmpty and T98GcGAS were transfected or not with biotinylated dsDNA prior to whole cell extraction and pull-down using streptavidin-affinity beads. Inputs and eluates were analyzed by WB using indicated antibodies.


J T98GcGAS were transfected or not with dsDNA in presence or not of NU7441 , prior to WB using indicated antibodies .

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J T98GcGAS were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.


K THP - 1 were transfected or not with dsDNA in presence or not of NU7441 , prior to WB using indicated antibodies .

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K THP-1 were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.


F T98GEmpty T98GcGAS stably expressing a GFP reporter ( T98G - GFPEmpty T98G - GFPcGAS , respectively ) were xenotransplanted into head of tg ( mfap4 : RFP ) zebrafish at 3 days post fertilization ( dpf ) . Zebrafish were imaged daily over 3 days . graph represents mean ( ± ) of growth normalized by on day of transplantation ( n = 21 T98G - GFPEmpty n = 29 T98G - GFPcGAS ) .

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F T98GEmpty and T98GcGAS stably expressing a GFP reporter (T98G-GFPEmpty and T98G-GFPcGAS, respectively) were xenotransplanted into the head of tg(mfap4:RFP) zebrafish line at 3 days post fertilization (dpf). Zebrafish embryos were imaged daily over 3 days. The graph represents the mean (±SEM) percentage of tumor growth normalized by the area on the day of transplantation (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).


A Graph represents mean ( ± ) fold migration of THP - 1 through a 3 µm transwell insert when conditioned media from T98GEmpty or T98GcGAS was applied to lower chamber for 6 h . CCL2 was used as positive control ( n = 8 biological replicates ) .

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A Graph represents the mean (± SEM) fold migration of THP-1 cells through a 3 µm transwell insert when conditioned media from T98GEmpty or T98GcGAS was applied to lower chamber for 6 h. CCL2 was used as positive control (n=8 biological replicates).


E Graph presents of macrophages recruited at site 24 h post xenotransplantation in D ( n = 21 T98G - GFPEmpty n = 29 T98G - GFPcGAS ) .

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E Graph presents the quantification of macrophages recruited at tumor site 24 h post xenotransplantation in D (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).


G Graph presents survival rate of glioblastoma from A that present PRKDClow / MB21D1low , PRKDChigh / MB21D1low PRKDChigh / MB21D1high expression .

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G Graph presents the survival rate of glioblastoma patients from A that present PRKDClow/MB21D1low, PRKDChigh/MB21D1low and PRKDChigh/MB21D1high expression.


H Violin plots present expression of PRKDC MB21D1 in datasets analyzed in A , on grade ( II to IV ) .

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H Violin plots present the expression of PRKDC and MB21D1 in datasets analyzed in A, based on tumor grade (II to IV).


3D - brain model , horizontal brain section illustrating transgenic human P301Ltau expression in entorhinal cortex ( green , EC ) of ECrTgTaumouse lines , propagation of transgenic tau to dentate gyrus ( DG ) . Tau composition in ECrTgTau control mouse lines investigated .

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3D-brain model, horizontal brain section illustrating transgenic human P301Ltau expression in the entorhinal cortex (green, EC) of the ECrTgTaumouse lines, and the propagation of transgenic tau to the dentate gyrus (DG). Tau composition in ECrTgTau and control mouse lines investigated.


Immunostained horizontal sections show expression of human P301Ltau in ECneurons in absence of endogenous mousetau ( ECrTgTau - Mapt0 / 0 ) . in situ hybridization of humantau mRNA combined with immunofluorescent labeling ( Immuno - FISH ) of humantau ( huTau ) verifies P301Ltau transgene expression in EC . Scale bars 50 μm .

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Immunostained horizontal sections show the expression of human P301Ltau in ECneurons in absence of endogenous mousetau (ECrTgTau-Mapt0/0). Fluorescence in situ hybridization of humantau mRNA combined with immunofluorescent labeling (Immuno-FISH) of humantau protein (huTau) verifies P301Ltau transgene expression in the EC. Scale bars 50 μm.


Propagation of humantau to neurons in DG ( arrows ) in ECrTgTau - Mapt0 / 0 mice . Close - ups show DG neurons from three ECrTgTau - Mapt0 / 0 mice ( DG I - III ) . Immuno - FISH proofs absence of humantau expression in DG neurons , which have huTau but no humantau mRNA . Scale bars 50 μm .

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Propagation of humantau protein to neurons in the DG (white arrows) in ECrTgTau-Mapt0/0 mice. Close-ups show DG neurons from three ECrTgTau-Mapt0/0 mice (DG I-III). Immuno-FISH proofs the absence of humantau expression in DG neurons, which have huTau protein but no humantau mRNA. Scale bars 50 μm.


Immunostained horizontal sections of ECrTgTaumice show expression of human P301Ltau in ECneurons in presence of endogenous mousetau . Close - ups show DGneurons from three ECrTgTaumice ( DG I - III ) Immuno - FISH proofs absence of humantau expression in these DGneurons . Scale bars 50 μm .

Immunostained horizontal sections of ECrTgTaumice show the expression of human P301Ltau in ECneurons in presence of endogenous mousetau. Close-ups show DGneurons from three ECrTgTaumice (DG I-III) and Immuno-FISH proofs the absence of humantau expression in these DGneurons. Scale bars 50 μm.


Human P301Ltau propagation to DG neurons ( arrows ) in presence of endogenous mouse tau in ECrTgTau mice . Scale bars 50 µm .

Human P301Ltau propagation to DG neurons (white arrows) in presence of endogenous mouse tau in ECrTgTau mice. Scale bars 50 µm.


Human ( huTau , antibody Tau13 ) total tau ( hu + moTau , DAKO ) in entorhinal cortex ( EC ) extracts from 18 - month - oldmice show equal human P301Ltau expression in ECrTgTau ECrTgTau - Mapt0 / 0 mice ( p = 0 . 201 , n = 3 mice / group , one - way ANOVA with Bonferroni correction )

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Human (huTau, antibody Tau13) and total tau (hu+moTau, DAKO) levels in entorhinal cortex (EC) extracts from 18-month-oldmice show equal human P301Ltau expression in ECrTgTau and ECrTgTau-Mapt0/0 mice (p=0.201, n=3 mice/group, one-way ANOVA with Bonferroni correction)


number of human tau - positive bodies in DG ( p = 0 . 58 , n = 4 sections 3 mice / group ) human tau in hippocampal ( HPC ) extracts ( p = 0 . 14 , n = 3 mice / group ) were similar in ECrTgTau - Mapt0 / 0 ECrTgTau mice ( two - Student ' s T - ) .

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The number of human tau-positive cell bodies in the DG (p=0.58, n=4 sections and 3 mice/group) and human tau in hippocampal (HPC) extracts (p=0.14, n=3 mice/group) were similar in ECrTgTau-Mapt0/0 and ECrTgTau mice (two-tailed Student's T-test).


cortical neuron cultures that were transduced with AAVeGFP - 2a - P301Ltau at 7 DIV , fixed immunostained for GFP humantau ( Tau13 antibody ) at 14 DIV , show tau donor ( GFP + , huTau + ; ~ 10 % neurons ) a small number of tau recipient neurons ( GFP - , huTau + ; ~ 1 % neurons ) . Western blot of whole lysates verified efficient cleavage ( ~ 95 % ) of eGFP P301Ltau by 2a peptide ( n = 3 ) .

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Primary cortical neuron cultures that were transduced with AAVeGFP-2a-P301Ltau at 7 DIV, and fixed and immunostained for GFP and humantau (Tau13 antibody) at 14 DIV, show tau donor (GFP+, huTau+; ~10% neurons) and a small number of tau recipient neurons (GFP-, huTau+; ~1% neurons). Western blot of whole cell lysates verified efficient cleavage (~95%) of eGFP and P301Ltau by the 2a peptide (n=3).


Eight after AAV injection into right EC of aged Mapt0 / 0 mice ( = 3 ) , immunostained brain sections showed that huTauP301L ( red ) propagated to a few DG " recipient neurons " ( arrows ) . Scale bar 50 µm .

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Eight weeks after AAV injection into right EC of aged Mapt0/0 mice (=3), immunostained brain sections showed that huTauP301L (red) propagated to a few DG "recipient neurons" (white arrows). Scale bar 50 µm.


Unilateral AAV - mediated human P301L tau expression in EC DG of - matched WT mice ( n = 3 ) . Representative of brain sections show donor neurons in injected EC DG , a few tau recipient neurons ( arrows ) adjacent to AAV injection site . Scale bar 50 µm . In hemisphere of same brain section , some tau recipient neurons ( arrows ) were also present in ( non - injected ) axonal projection areas in EC ( GFP - filled terminal ends ) . Scale bar 100 µm .

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Unilateral AAV-mediated human P301L tau expression in the EC and DG of age-matched WT mice (n=3). Representative images of brain sections show donor neurons in the injected EC and DG, and a few tau recipient neurons (white arrows) adjacent to the AAV injection site. Scale bar 50 µm.In the contralateral hemisphere of the same brain section, some tau recipient neurons (white arrows) were also present in the (non-injected) axonal projection areas in the contralateral EC (GFP-filled terminal ends). Scale bar 100 µm.


A . Brain sections from 18 - month - old ECrTgTau - Mapt0 / 0 ECrTgTau mice were co - immuolabeled for human tau misfolded tau ( Alz50 ) . Misfolded tau was only found in EC DG neurons ( arrows ) of ECrTgTau but not ECrTgTau - Mapt0 / 0 ( n = 4 sections / mouse , 3 mice / group ) . Scale bars 50 μm .

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A. Brain sections from 18-month-old ECrTgTau-Mapt0/0 and ECrTgTau mice were co-immuolabeled for human tau and misfolded tau (Alz50). Misfolded tau was only found in EC and DG neurons (white arrows) of ECrTgTau but not ECrTgTau-Mapt0/0 animals (n=4 sections/mouse, 3 mice/group). Scale bars 50 μm.


B + C . Immunofluorescence labeling stereological counting of microglia in entorhinal cortex ( B ) astrocytes in hippocampus ( C ) indicated early signs of neurodegeneration in ECrTgTaumice . significantly increased number of Iba1 - positive microglia in EC layer II / III of ECrTgtau mice ( compared to WT ) was partially rescued in ECrTgTau - Mapt0 / 0 mice ( non - significant ) . number of GFAP - positive astrocytes was similar across genotypes ( non - significant ) . Mean ± , n = 4 sections per mouse , 3 mice / group , one - way ANOVA with Bonferroni correction . Scale bars 100 μm .

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B+C. Immunofluorescence labeling and stereological counting of microglia in entorhinal cortex (B) and astrocytes in hippocampus (C) indicated early signs of neurodegeneration in ECrTgTaumice. The significantly increased number of Iba1-positive microglia in the EC layer II/III of ECrTgtau mice (compared to WT) was partially rescued in ECrTgTau-Mapt0/0 mice (non-significant). The number of GFAP-positive astrocytes was similar across all genotypes (non-significant). Mean±SEM, n=4 sections per mouse, 3 mice/group, one-way ANOVA with Bonferroni correction. Scale bars 100 μm.


Human , mouse total tau in cortical TBS - extracts of rTg4510 , rTg4510 - Mapt0 / 0 , control mice : amount of human tau ( Tau13 antibody ) was comparable in rTg4510 rTg4510 - Mapt0 / 0 , moTau ( Tau / 5 ) was comparable in WT rTg4510 , total tau ( hu + moTau , DAKO antibody ) were ( expected ) highest in rTg4510 mice . Mean ± , n = 3 mice / group , non - significant .

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Human, mouse and total tau protein levels in cortical TBS-extracts of rTg4510, rTg4510-Mapt0/0, and control mice: The amount of human tau (Tau13 antibody) was comparable in rTg4510 and rTg4510-Mapt0/0, moTau (Tau/5) was comparable in WT and rTg4510, and total tau levels (hu+moTau, DAKO antibody) were (expected) highest in rTg4510 mice. Mean±SEM, n=3 mice/group, non-significant.


Whole brain weights of 9 - month - old revealed pronounced brain matter loss in rTg4510 compared to WT mice ( loss > 16 % ) , which was rescued in rTg4510 - Mapt0 / 0 mice to > 96 % . Mean ± , n = 5 mice / group .

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Whole brain weights of 9-month-old animals revealed pronounced brain matter loss in rTg4510 compared to WT mice (weight loss >16%), which was rescued in rTg4510-Mapt0/0 mice to >96%. Mean±SEM, n=5 mice/group.


Cortical thickness measured adjacent to HPC , from CTX surface to corpus callosum , was decreased in rTg4510 mice by ≈ 25 % compared to WT mice . rTg4510 - Mapt0 / 0 showed no CTX thinning compared to Mapt0 / 0 or WT mcie . Mean ± , n = 3 mice / group .

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Cortical thickness measured adjacent to HPC, from CTX surface to corpus callosum, was decreased in rTg4510 mice by ≈25% compared to WT mice. rTg4510-Mapt0/0 showed no CTX thinning compared to Mapt0/0 or WT mcie. Mean±SEM, n=3 mice/group.


number of neurons ( NeuN + ) in cortex of rTg4510 mice was significantly reduced to ≈ 67 % compared to both WT rTg4510 - Mapt0 / 0 . Mean ± , n = 3 mice / group .

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The number of neurons (NeuN+ cells) in the cortex of rTg4510 mice was significantly reduced to ≈67% compared to both WT and rTg4510-Mapt0/0. Mean±SEM, n=3 mice/group.


volume of hippocampal region CA1 , with CT most effected regions in rTg4510 mice , was significantly reduced in rTg4510 by ~ 70 % volume ; rTg4510 - Mapt0 / 0 had significantly larger CA1 volume left ( reduced by only ~ 40 % ) . Mean ± , n = 3 mice / group .

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The volume of hippocampal region CA1, with CT the most effected regions in rTg4510 mice, was significantly reduced in rTg4510 by ~70% volume; rTg4510-Mapt0/0 had significantly larger CA1 volume left (reduced by only ~40%). Mean±SEM, n=3 mice/group.


rTg4510 showed strong signs of neuroinflammation with extremely high numbers of activated astroglia ( GFAP + , red ) microglia ( Iba1 + , ) in CTX compared to WT mice . Both astro - microgliosis was reduced by ~ 50 % in rTg4510 - Mapt0 / 0 mice . Mean ± , n = 3 sections / mouse 5 mice per / group . For panels : two - Student ' s T - one - way ANOVA with Bonferroni for multiple comparison , ns not significant , scale bars 100 µm .

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rTg4510 showed strong signs of neuroinflammation with extremely high numbers of activated astroglia (GFAP+, red) and microglia (Iba1+, white) in the CTX compared to WT mice. Both astro- and microgliosis was reduced by ~50% in rTg4510-Mapt0/0 mice. Mean±SEM, n=3 sections/mouse and 5 mice per/group. For all panels: two-tailed Student's T-test and one-way ANOVA with Bonferroni for multiple comparison, ns not significant, scale bars 100 µm.


Cortical extracts from rTg4510 - Mapt0 / 0 brains had significantly less phospho - tau ( CP13 , PHF1 , 12E8 ) than rTg4510 extracts . Compared to WT mice , both transgenic tau lines had high of phospho - tau . Mean ± , n = 3 mice / group .

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Cortical extracts from rTg4510-Mapt0/0 brains had significantly less phospho-tau (CP13, PHF1, 12E8) than rTg4510 extracts. Compared to WT mice, both transgenic tau lines had high levels of phospho-tau. Mean±SEM, n=3 mice/group.


Representative of gallyas silver stained aggregated tau in cortices from 12 - month - old mice unravel stunning differences in degree of tau pathology in rTg4510 compared to rTg4510 - Mapt0 / 0 mice . Mean ± , n = 3 mice / group .

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Representative images of gallyas silver stained aggregated tau in cortices from 12-month-old mice unravel stunning differences in the degree of tau pathology in rTg4510 compared to rTg4510-Mapt0/0 mice. Mean±SEM, n=3 mice/group.


Higher magnification of silver ( 12 month - old ) Thioflavine - S ( 9month - old ) stained cortices show mature tangles ( arrows in Thio - S stain ) in rTg4510 rTg4510 - Mapt0 / 0 mice ; enhanced pathological changes such as neuritic tau accumulation neuropil vacuolation around NFTs are found only in rTg4510 mice . Stereological counting revealed similar numbers of cortical NFTs between in rTg4510 rTg4510 - Mapt0 / 0 mice at 9 12 month of . Because of pronounced neuronal only in rTg4510 mice , of tangle - bearing neurons was ≈ 1 . 6 to 1 . 8 - fold higher higher in 9 12 - month old rTg4510 mice . Mean ± , n = 3 sections / mouse , 3 mice / group .

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Higher magnification images of silver (12 month-old) and Thioflavine-S (9month-old) stained cortices show mature tangles (white arrows in Thio-S stain) in rTg4510 and rTg4510-Mapt0/0 mice; enhanced pathological changes such as neuritic tau accumulation and neuropil vacuolation around NFTs are found only in rTg4510 mice. Stereological counting revealed similar numbers of cortical NFTs between in rTg4510 and rTg4510-Mapt0/0 mice at 9 and 12 month of age. Because of the pronounced neuronal death only in rTg4510 mice, the percentage of tangle-bearing neurons was ≈1.6 to 1.8-fold higher higher in 9 and 12-month old rTg4510 mice. Mean±SEM, n=3 sections/mouse, 3 mice/group.


Immuno - FISH for huTau mRNA ( green ) phospho - tau ( PHF1 , red ) shows obvious differences in distribution of neurons in cortex layer II / III : 9 - month - old rTg4510 mice had more neurons filled with NFT - like phosphorylated tau ( PHF1 + , red ) , rTg4510 - Mapt0 / 0 mice had significantly more huTau mRNA positive neurons ( FISH + ) . rTg4510 - Mapt0 / 0 mice had also more neurons still expressing both PHF1 huTau mRNA ( PHF1 + FISH + ) , suggesting a reduced neurotoxicity of P301Ltau expression in rTg4510 - Mapt0 / 0 mice . Mean ± , n = 3 sections per mouse , 3 mice / group . For panels : two - Student ' s T - one - way ANOVA with Bonferroni for multiple comparison , ns not significant , scale bars 100 μm .

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Immuno-FISH for huTau mRNA (green) and phospho-tau (PHF1, red) shows obvious differences in the distribution of neurons in cortex layer II/III: 9-month-old rTg4510 mice had more neurons filled with NFT-like phosphorylated tau (PHF1+, red), and rTg4510-Mapt0/0 mice had significantly more huTau mRNA positive neurons (FISH+). rTg4510-Mapt0/0 mice had also more neurons still expressing both PHF1 and huTau mRNA (PHF1+ FISH+), suggesting a reduced neurotoxicity of P301Ltau expression in rTg4510-Mapt0/0 mice. Mean±SEM, n=3 sections per mouse, 3 mice/group. For all panels: two-tailed Student's T-test and one-way ANOVA with Bonferroni for multiple comparison, ns not significant, scale bars 100 μm.


Immuno - FISH showing EC neurons having both misfolded somatic tau ( Alz50 , red ) human tau mRNA ( green ; arrows ) in rTg4510 - Mapt0 / 0 but rarely in rTg4510 mice . n = 3 sections , 2 mice / group . Scale bars 50 μm .

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Immuno-FISH showing EC neurons having both misfolded somatic tau (Alz50, red) and human tau mRNA (green; white arrows) in rTg4510-Mapt0/0 but rarely in rTg4510 mice. n=3 sections, 2 mice/group. Scale bars 50 μm.


Extraction of cortices revealed similar human tau ( Tau13 ) in TBS - extracts ( not significant ) but significantly more human tau in TritonX - 100 ( TX - 100 ) extracts of rTg4510 - Mapt0 / 0 compared to rTg4510 mice . Mean ± , n = 3 mice / group , two - Student ' s T , ns non significant .

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Extraction of cortices revealed similar human tau (Tau13) in TBS-extracts (not significant) but significantly more human tau in TritonX-100 (TX-100) extracts of rTg4510-Mapt0/0 compared to rTg4510 mice. Mean±SEM, n =3 mice/group, two-tailed Student's T test, ns non significant.


Native gel electrophoresis of cortical TBS - extracts showed small differences in HMW ( oligomeric ) human tau between rTg4510 rTg4510 - Mapt0 / 0 brains . Western blot lanes were averaged across ~ 2 / 3 of width ( black rectangular arrow in Tau13 blot ) . mean ± ( n = 3 mice / group ) of these averages was plotted as longitudinal lane profiles . Differences in HMW tau are indicated by red black arrows .

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Native gel electrophoresis of cortical TBS-extracts showed small differences in HMW (oligomeric) human tau between rTg4510 and rTg4510-Mapt0/0 brains. Western blot lanes were averaged across ~2/3 of the width (black rectangular and arrow in Tau13 blot). The mean±SEM (n=3 mice/group) of these averages was plotted as longitudinal lane profiles. Differences in HMW tau are indicated by red and black arrows.


TBS - brain extracts were applied to a HEK293 tau aggregation seeding ( Holmes et al , 2014 , Sanders et al , 2014 ) , in which TauRDP301S - CFP TauRDP301S - YFP are co - expressed intracellularly . formation of intracellular TauRDP301S leads to Foerster resonance energy transfer ( FRET ) activity between co - aggregated CFP YFP - tags correlates with tau aggregation seeding activity of applied brain extracts . After 24 hours , treated with extract ( 0 . 5 µg 1 . 0 µg total per 96 well ) from 9 - month - old rTg4510 had significantly more intracellular YFP - positive ( arrows ) compared to rTg4510 - Mapt0 / 0 extracts ; FRET activity of TauRDP301S appeared similar for both rTg4510 rTg4510 - Mapt0 / 0 . WT Mapt0 / 0 extracts never showed seeding activity . Addition of lipofectamine corrected for differences in uptake of tau led to similar differences in seeding activity between rTg4510 rTg4510 - Mapt0 / 0 mice . Two - Student ' s T - , mean ± ns , not significant , Insets 100 µm , Scale bars 50 µm .

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TBS-brain extracts were applied to a HEK293 cell tau aggregation seeding assay(Holmes et al, 2014, Sanders et al, 2014), in which TauRDP301S-CFP and TauRDP301S-YFP are co-expressed intracellularly. The formation of intracellular fluorescent TauRDP301S aggregates leads to Foerster resonance energy transfer (FRET) activity between co-aggregated CFP and YFP-tags and correlates with the tau aggregation seeding activity of the applied brain extracts. After 24 hours, cells treated with extract (0.5 µg and 1.0 µg total protein per 96 well) from 9-month-old rTg4510 had significantly more intracellular YFP-positive (white arrows) aggregates compared to rTg4510-Mapt0/0 extracts; FRET activity of TauRDP301S aggregates appeared similar for both rTg4510 and rTg4510-Mapt0/0. WT and Mapt0/0 extracts never showed seeding activity. Addition of lipofectamine corrected for differences in cellular uptake of tau and led to similar differences in seeding activity between rTg4510 and rTg4510-Mapt0/0 mice. Two-tailed Student's T-test, mean±SEM ns, not significant, Insets 100 µm, Scale bars 50 µm.


A - D LSM of proximity ligation ( PLA ) dots composed of VEGFR3 phosphorylated tyrosine ( p - Tyr ) on cross - sections through jugular lymph sac / primordial thoracic ( jls / pTD ) of E13 . 5 control ILK K . O . . Scale bars : 10 µm . E - H LSM of PLA dots composed of VEGFR2 phosphorylated tyrosine ( p - Tyr ) on cross - sections through jls / pTD of E13 . 5 control ILK K . O . . Scale bars : 10 µm . I of PLA dots indicating VEGFR3 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK K . O . ( n = 9 per genotype ) , * P = 0 . 022 . J of PLA dots indicating VEGFR2 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK K . O . ( n = 3 per genotype ) . K of total number of PLA dots indicating both VEGFR2 ( left ) VEGFR3 ( right ) with phosphorylated tyrosine ( p - Tyr ) per jls / pTD section of E13 . 5 control or ILK K . O . ( n = 3 per genotype ) , * P = 0 . 005 ( VEGFR2 / p - Tyr in ILK K . O . versus VEGFR3 / p - Tyr in ILK K . O . ) , * P = 0 . 013 ( VEGFR3 / p - Tyr in control versus VEGFR3 / p - Tyr in ILK K . O . )

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A-D LSM images of proximity ligation assay (PLA) dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. E-H LSM images of PLA dots composed of VEGFR2 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. I Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 9 embryos per genotype), *P = 0.022. J Quantification of the PLA dots indicating VEGFR2 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype). K Quantification of the total number of PLA dots indicating both VEGFR2 (left) and VEGFR3 (right) with phosphorylated tyrosine (p-Tyr) per jls/pTD section of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype), *P = 0.005 (VEGFR2/p-Tyr in ILK K.O. versus VEGFR3/p-Tyr in ILK K.O.), *P = 0.013 (VEGFR3/p-Tyr in control versus VEGFR3/p-Tyr in ILK K.O.)


LSM of cross - sections through jugular lymph sac / primordial thoracic ( jls / pTD ) of an E13 . 5 control stained for surface VEGFR3 β1 integrin . Scale bar : 10

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LSM images of cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of an E13.5 control embryo stained for surface VEGFR3 and β1 integrin. Scale bar: 10 µm


LSM of proximity ligation ( PLA ) dots ( arrows ) composed of VEGFR3 β1 integrin on cross - sections through jls / pTD of E13 . 5 control ILK K . O . . Scale bars : 10 µm of VEGFR3 / β1 integrin PLA dots normalised to Lyve1 - positive of control or ILK K . O . ( n = 6 per genotype ) ,

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LSM images of proximity ligation assay (PLA) dots (arrows) composed of VEGFR3 and β1 integrin on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots normalised to Lyve1-positive area of control or ILK K.O. embryos (n = 6 embryos per genotype),


Bright - field image of an E13 . 5 Flk1 - Cre ; Ilk ∆ / + ; Itgb1 ∆ / + mouse ( referred to as " Control " ) with a heterozygous deletion of both Ilk Itgb1 in endothelial , a LSM image of a stained cross - section through its jugular lymph sac / primordial thoracic ( jls / pTD ) . Scale bars : 500 100 µm , respectively . C , D Bright - field image of an E13 . 5 Flk1 - Cre ; Ilk ∆ / ∆ ; Itgb1 ∆ / + ( referred to as " ILK & β1 integrin K . O . " ) with a homozygous deletion of Ilk heterozygous deletion of Itgb1 in endothelial , a LSM image of a stained cross - section through its jls / pTD . Scale bars : 500 100 µm , respectively . E - H LSM of cross - sections through jls / pTD of E13 . 5 control ILK & β1 integrin K . O . stained for proliferation marker phospho - Histone H3 . Arrows point to phospho - Histone H3 - positive

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Bright-field image of an E13.5 Flk1-Cre;Ilk∆/+;Itgb1∆/+ mouse embryo (referred to as "Control") with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jugular lymph sac / primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 µm, respectively. C, D Bright-field image of an E13.5 Flk1-Cre;Ilk∆/∆;Itgb1∆/+ embryo (referred to as "ILK & β1 integrin K.O.") with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jls/pTD. Scale bars: 500 and 100 µm, respectively. E-H LSM images of cross-sections through the jls/pTD of E13.5 control and ILK & β1 integrin K.O. embryos stained for the proliferation marker phospho-Histone H3. Arrows point to phospho-Histone H3-positive LEC


LSM of PLA dots composed of VEGFR3 phosphorylated tyrosine ( p - Tyr ) on stained cross - sections through jls / pTD of control ILK & β1 integrin K . O . . Arrows point to PLA dots within Lyve1 stained . Scale bars : 10

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LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on stained cross-sections through the jls/pTD of control and ILK & β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1 stained area. Scale bars: 10 µm


Number of LECs per jls / pTD section in E13 . 5 control or ILK & β1 integrin K . O .

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Number of LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryos


LEC proliferation as determined by number of phospho - Histone H3 - positive LECs per jls / pTD section in E13 . 5 control or ILK & β1 integrin K . O .

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LEC proliferation as determined by the number of phospho-Histone H3-positive LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryos


of PLA dots indicating VEGFR3 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK & β1 integrin K . O .

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Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK & β1 integrin K.O. embryos


LSM of a whole mount stained adult Prox1 - CreERT2 ; Ilk + / + mouse ear ( referred to as " Adult Control " ) Prox1 - CreERT2 ; Ilk ∆ / ∆ mouse ear ( referred to as " Adult ILK K . O . " ) , with higher magnification indicated by dashed lines . Scale bars : 500 LSM of a whole - mount stained adult control ILK K . O . ear , with higher magnification indicated by dashed lines . Arrows point to proliferating LECs . Scale bars : 100 20 µm ,

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LSM images of a whole mount stained adult Prox1-CreERT2;Ilk+/+ mouse ear (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse ear (referred to as "Adult ILK K.O."), with higher magnification images indicated by dashed lines. Scale bars: 500 and LSM images of a whole-mount stained adult control and ILK K.O. ear, with higher magnification images indicated by dashed lines. Arrows point to proliferating LECs. Scale bars: 100 and 20 µm, respectively


Dermal lymph vessel density as determined by VEGFR3 - positive normalised to total analysed of adult control or ILK K . O . mice ( n = 4 control n = 5 ILK K . O . mice ) ,

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Dermal lymph vessel density as determined by VEGFR3-positive area normalised to the total analysed area of adult control or ILK K.O. mice (n = 4 control and n = 5 ILK K.O. mice),


LEC proliferation as determined by phospho - Histone H3 - positive LECs normalised to analysed VEGFR3 - positive in skin of adult control or ILK K . O . mice ( with n = 7 control n = 5 ILK K . O . mice ) ,

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LEC proliferation as determined by phospho-Histone H3-positive LECs normalised to analysed VEGFR3-positive area in the skin of adult control or ILK K.O. mice (with n = 7 control and n = 5 ILK K.O. mice),


VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K . O . mice ( n = 3 mice per genotype )

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VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K.O. mice (n = 3 mice per genotype)


LSM of a whole mount stained adult control ILK K . O . cornea , with detailed on their right ( L " , M " ) . Arrows point to lymph vessels protruding into cornea ( encircled by a dotted ) . Scale bars : 500 µm 100 µm , re Number of lymph vessels in control or ILK K . O . corneas ( n = 5 n = 7 corneas ) ,

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LSM images of a whole mount stained adult control and ILK K.O. cornea, with detailed images on their right (L", M"). Arrows point to lymph vessels protruding into the cornea (encircled by a dotted line). Scale bars: 500 µm and 100 µm, re Number of lymph vessels in control or ILK K.O. corneas (n = 5 and n = 7 corneas),


LSM of cross - sections through heart of an adult Prox1 - CreERT2 ; Ilk + / + mouse ( referred to as " Adult Control " ) Prox1 - CreERT2 ; Ilk ∆ / ∆ mouse ( referred to as " Adult ILK K . O . " ) showing outer lateral region of left ventricle ( LV ) Overall cardiac lymph vessel blood vessel density in heart sections as determined by Lyve1 - positive CD31 - positive ( normalised to total analysed myocardial ) , respectively ( n = 4 control n = 5 ILK K . O . mice )

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LSM images of cross-sections through the heart of an adult Prox1-CreERT2;Ilk+/+ mouse (referred to as "Adult Control") and Prox1-CreERT2;Ilk∆/∆ mouse (referred to as "Adult ILK K.O.") showing the outer lateral region of the left ventricle (LV) Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively (n = 4 control and n = 5 ILK K.O. mice)


LSM of cross - sections through heart of an adult control ILK K . O . mouse four after myocardial ischemia reperfusion ( MI / R ) , showing outer lateral region of LV . Scale bars : 100 µm Overall cardiac lymph vessel blood vessel density in heart sections as determined by Lyve1 - positive CD31 - positive ( normalised to total analysed myocardial ) , respectively . ( n = 5 control n = 7 ILK K . O . mice ) , P = 0 . 051 ( cardiac lymph vessel density in adult control versus adult ILK K . O . ) , * P = 0 . 025 ( cardiac blood vessel density in adult control versus adult ILK K . O . )

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LSM images of cross-sections through the heart of an adult control and ILK K.O. mouse four weeks after myocardial ischemia and reperfusion (MI/R), showing the outer lateral region of the LV. Scale bars: 100 µm Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively. (n = 5 control and n = 7 ILK K.O. mice), P = 0.051 (cardiac lymph vessel density in adult control versus adult ILK K.O.), *P = 0.025 (cardiac blood vessel density in adult control versus adult ILK K.O.)


of adult human LECs after 1 hour of BrdU incorporation previous transfections with control or ILK siRNA . Scale bars : 50

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Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 µm


LEC proliferation as determined by number of BrdU - positive normalised to total number of LECs previously transfected with control siRNA or ILK siRNAs in presence of VEGF - C Cys156Ser ( n = 3 independent transfections per siRNA ) , * P = 0 . 032 ( control versus ILK - 1 ) , * P = 0 . 005 ( control versus ILK - 2 ) , * P = 0 . 0003 ( control versus ILK - 3 )

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LEC proliferation as determined by the number of BrdU-positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 3 independent transfections per siRNA), *P = 0.032 (control versus ILK-1), *P = 0.005 (control versus ILK-2), *P = 0.0003 (control versus ILK-3)


VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in presence of VEGF - C Cys156Ser ( n = 4 ( control siRNA , ILK - 1 siRNA , ILK - 3 siRNA ) , or n = 8 ( ILK - 2 siRNA ) independent transfections per siRNA ) , * P = 0 . 0001 ( control versus each siRNA )

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VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 4 (control siRNA, ILK-1 siRNA, and ILK-3 siRNA), or n = 8 (ILK-2 siRNA) independent transfections per siRNA), *P = 0.0001 (control versus each siRNA)


LSM of VEGFR3 / β1 integrin PLA dots in human LECs transfected with control or ILK siRNA . Scale bars : 10 µm of VEGFR3 / β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA ) , P = 0 . 234 ( control versus ILK - 1 ) , * P = 0 . 024 ( control versus ILK - 2 ) , * P = 0 . 001 ( control versus ILK - 3 )

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LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs (n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK-1), *P = 0.024 (control versus ILK-2), *P = 0.001 (control versus ILK-3)


LSM of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes . Red dots are PLA dots composed of VEGFR3 β1 integrin . Scale bars : 10 µm of VEGFR3 / β1 integrin PLA dots per human LEC with ( + ) or without ( - ) mechanical stretch ( n = 6 independent stretch chambers ) ,

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LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (-) mechanical stretch (n = 6 independent stretch chambers),


Western blot ( WB ) image of human LECs that were either kept unstretched or stretched for 30 minutes , used for immunoprecipitation ( IP ) of HA - tagged β1 integrin from whole lysates with subsequent detection of interacting ILK in IP lysates of ILK amount in IP lysates from LECs with ( + ) or without ( - ) mechanical stretch ; normalised to respective amount of HA - tagged β1 integrin ( n = 3 ( unstretched ) or n = 5 ( stretched ) independent stretch

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Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 minutes, and used for immunoprecipitation (IP) of HA-tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (-) mechanical stretch; normalised to the respective amount of HA-tagged β1 integrin (n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers


Confocal of HeLa stained with NF45 / NF90 antibodies antibodies against nucleolin fibrillarin . Scale bar , 10 μm .

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Confocal images of HeLa cells stained with NF45/NF90 antibodies and antibodies against nucleolin and fibrillarin. Scale bar, 10 μm.


HEK293 were infected with lentiviruses containing negative control shRNA ( shNC ) or NF45 / NF90 specific shRNA ( shNF45 # 1 , shNF45 # 2 , shNF90 # 1 shNF90 # 2 ) for 24 h , then transfected with rDNA - Luc . Luciferase activity was measured after a further 24 h ( n = 3 ) . Western blots show of NF45 NF90 . Empty vector ( EV ) , FLAG - NF45 plasmid ( NF45 ) or FLAG - NF90 plasmid ( NF90 ) were transfected into HEK293 for 24 h , then rDNA - Luc was co - transfected into . Luciferase activity was measured after a further 24 h ( n = 3 ) . Western blots show of FLAG - NF45 FLAG - NF90 .

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HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1 and shNF90#2) for 24 h, and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90.   Empty vector (EV), FLAG-NF45 plasmid (NF45) or FLAG-NF90 plasmid (NF90) were transfected into HEK293 cells for 24 h, and then rDNA-Luc was co-transfected into the cells. Luciferase activity was measured after a further 24 h (n=3). Western blots show the protein levels of FLAG-NF45 and FLAG-NF90.


HEK293 were infected with lentiviruses containing negative control shRNA ( shNC ) or NF45 / NF90 specific shRNA ( shNF45 # 1 , shNF45 # 2 , shNF90 # 1 shNF90 # 2 ) for 24 h , then transfected with rDNA - Luc . Luciferase activity was measured after a further 24 h ( n = 3 ) . Western blots show of NF45 NF90 .

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HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1 and shNF90#2) for 24 h, and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90.


E - F qPCR of 45S pre - rRNA from HEK293 treated with indicated shRNAs ( E ) plasmids ( F ) for 24 h . Western blots show expression of NF45 , NF90 , FLAG - NF45 FLAG - NF90 . ( n = 3 )

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E-F The qPCR analysis of 45S pre-rRNA from HEK293 cells treated with indicated shRNAs (E) and plasmids (F) for 24 h. Western blots show the expression levels of NF45, NF90, FLAG-NF45 and FLAG-NF90. (n = 3)


In Jurkat , a ChIP was performed with control IgG , NF45 NF90 antibodies , then precipitated was analyzed using qPCR with aforementioned primers . relative rDNA fold - enrichment was normalized to control IgG treatment . ( n = 3 )

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In Jurkat cells, a ChIP assay was performed with control IgG, NF45 and NF90 antibodies, and then the precipitated DNA was analyzed using qPCR with the aforementioned primers. The relative rDNA fold-enrichment was normalized to control IgG treatment. (n=3)


EMSA . Lane 1 ) Biotin - labled rDNA probe . Lane 2 ) Biotin - labled rDNA probe + . Lane 3 ) Biotin - labled rDNA probe + + 100 - fold molar excess of biotin - unlabeled specific competitor . Lane 4 ) Biotin - labled rDNA probe + + 100 - fold molar excess of biotin - unlabeled mutant competitor . Lane 5 ) Biotin - labled rDNA probe + + NF90 antibody . Lane 6 ) Biotin - labled rDNA probe + + NF45 antibody . details were described in Materials Methods section .

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EMSA assay. Lane 1) Biotin-labled rDNA probe. Lane 2) Biotin-labled rDNA probe + nuclear protein. Lane 3) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled specific competitor. Lane 4) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled mutant competitor. Lane 5) Biotin-labled rDNA probe + nuclear protein + NF90 antibody. Lane 6) Biotin-labled rDNA probe + nuclear protein + NF45 antibody. The details were described in Materials and Methods section.


relative luciferase activity of HEK293T transfected with empty vector ( EV ) or FLAG - NF90 ( NF90 ) in combination with rDNA - Luc wild type ( WT ) or mutated rDNA - Luc reporter plasmid ( Mutant ) for 24 h . ( n = 3 ) Western blot shows FLAG - NF90 level .

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The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA -Luc wild type (WT) or mutated rDNA-Luc reporter plasmid (Mutant) for 24 h. (n=3) Western blot shows FLAG-NF90 protein level.


relative luciferase activity of HEK293T transfected with empty vector ( EV ) or FLAG - NF90 ( NF90 ) in combination with

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The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA


Co - IP of nucleolar with NF90 in HEK293 transfected with EV , following plasmids : GFP - S5 , GFP - L9 , GFP - B23 , GFP - UBF , GFP - C23 . * represents target .

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Co-IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV, and the following plasmids: GFP-S5, GFP-L9, GFP-B23, GFP-UBF, GFP-C23. * represents the target protein.


Co - IP of endogenous UBF with NF45 NF90 in HEK293 .

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Co-IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells.


HEK293 were infected by lentivirus containing NF45 - specfic shRNA or NF90 - specfic shRNA , then stimulated in serum for 0 , 3 6 h . lysates were prepared to analyze expression of indicated by western blot .

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HEK293 cells were infected by lentivirus containing NF45-specfic shRNA or NF90-specfic shRNA, and then stimulated in serum for 0, 3 and 6 h. Cell lysates were prepared to analyze the expression levels of the indicated proteins by western blot.


ChIP was used to determinate binding ability of NF45 NF90 to rDNA promoters in HeLa stimulated with serum for 0 , 3 6 h . ( n = 3 )

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ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0, 3 and 6 h. (n = 3)


ChIP of NF45 NF90 binding to H42 . 9 loci in HeLa with treatment of CX5461 or DMSO for 2 h . ( n = 3 )

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ChIP analysis of NF45 and NF90 binding to the H42.9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h. (n = 3)


Confocal of HeLa treated with CX5461 for 2 h showing NF45 NF90 colocalized with fibrillarin . Scale bar , 10 μm .

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Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm.


ChIP of NF45 NF90 binding to H42 . 9 loci in CD3 + T stimulated with P / I for 6 h . ( n = 3 )

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ChIP analysis of NF45 and NF90 binding to the H42.9 loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)


qPCR of pre - rRNA in CD3 + T treated with P / I for 6 h . ( n = 3 )

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qPCR analysis of pre-rRNA in CD3+ T cells treated with P/I for 6 h. (n=3)


CD3 + T were first silenced with shNC , shNF45 or shNF90 , then treated with P / I to extract total for qPCR of pre - rRNA . ( n = 3 )

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CD3+ T cells were first silenced with shNC, shNF45 or shNF90, and then treated with P/I to extract total RNA for qPCR analysis of pre-rRNA. (n=3)


of rDNA - luc activity in Jurkat with or without NF90 silence upon P / I stimulation or not . ( n = 3 )

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The analysis of rDNA-luc activity in Jurkat cells with or without NF90 silence upon P/I stimulation or not. (n=3)


Confocal of Jurkat to show NF45 ( left panel ) or NF90 ( right panel ) ( green ) colocalized with NFATc2 ( red ) in nucleoli . Scale bar , 10 μm .

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Confocal images of Jurkat cells to show NF45 (left panel) or NF90 (right panel) (green fluorescence) colocalized with NFATc2 (red fluorescence) in nucleoli. Scale bar, 10 μm.


Co - IP of NFATc2 NFATc1 with NF90 in Jurkat treated with P / I .

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Co-IP analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P/I.


ChIP of NFATc2 binding to rDNA loci in CD3 + T stimulated with P / I for 6 h . ( n = 3 )

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ChIP analysis of NFATc2 binding to the rDNA loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)


Jurkat co - transfected with rDNA - luc ( wild type or mutant plasmid ) with NF90 / or NFATc2 for 24 h , then rDNA - luc activity was detected after P / I sitmulation or not ( DMSO ) . ( n = 3 )

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Jurkat cells co-transfected with rDNA-luc (wild type or mutant plasmid) with NF90 and/or NFATc2 for 24 h, then rDNA-luc activity was detected after P/I sitmulation or not (DMSO). (n=3)


ChIP of NFATc2 binding to H42 . 9 loci in NF90 - slienced CD3 + T stimulated with P / I for 6 h . ( n = 3 )

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ChIP analysis of NFATc2 binding to the H42.9 loci in NF90-slienced CD3+ T cells stimulated with P/I for 6 h. (n=3)


CD69 expression , an early marker of lymphocyte activation , in CD3 + T infected with shNC , shNF45 or shNF90 lentivirus upon stimulation with P / I for 12 h .

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CD69 expression, an early marker of lymphocyte activation, in CD3+ T cells infected with shNC, shNF45 or shNF90 lentivirus upon stimulation with P/I for 12 h.


B - C PBMC with shNC , shNF45 or shNF90 lentivirus infection were stimulated with P / I for 12h , then freshly replaced medium without P / I after another 24 h culture was harvested for IL - 2 IFN - γ detections by ELISA . ( n = 3 )

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B-C PBMC cells with shNC, shNF45 or shNF90 lentivirus infection were stimulated with P/I for 12h, then the freshly replaced medium without P/I after another 24 h culture was harvested for IL-2 and IFN-γ detections by ELISA.(n=3)


proliferation of shNC , shNF45 or shNF90 - infected PBMCs was monitored using a CCK - 8 . ( n = 3 )

The proliferation of shNC, shNF45 or shNF90-infected PBMCs was monitored using a CCK-8 assay. (n = 3)


Confocal to show subcellular localization of NFATc2 in CD3 + T treated with P / I FK506 or CX5461 . Scale bar , 10 μm .

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Confocal images to show the subcellular localization of NFATc2 in CD3+ T cells treated with P/I and FK506 or CX5461. Scale bar, 10 μm.


Flow cytometry of CD69 expression in P / I - stimulated CD3 + T treated with or without FK506 or CX5461 .

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Flow cytometry analysis of CD69 expression in P/I-stimulated CD3+ T cells treated with or without FK506 or CX5461.


G - H ELISA assays of IL - 2 IFN - γ in CD3 + T with indicated treatments . ( n = 3 )

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G-H ELISA assays of IL-2 and IFN-γ in CD3+ T cells with the indicated treatments. (n = 3)


Flow cytometry of CFSE - labeled CD3 + T in presence of DMSO , FK506 CX5461 for 0 , 3 7 d .

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Flow cytometry analysis of CFSE-labeled CD3+ T cells in the presence of DMSO, FK506 and CX5461 for 0, 3 and 7 d.


qPCR of pre - rRNA expression from skin - heart - transplanted mouse spleens . ( n = 5 )

qPCR analysis of pre-rRNA expression from skin- and heart-transplanted mouse spleens. (n = 5)


pre - rRNA expression of PBMC from renal transplantation . ( Stable group , n = 7 . ABMR group , n = 8 . TCMR group , n = 8 ) .

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pre-rRNA expression levels of PBMC from renal transplantation patients. (Stable group, n = 7. ABMR group, n = 8. TCMR group, n = 8).


Skin graft survival curve in mice treated with DMSO , 2 . 0 mg / kg FK506 or 0 . 5 - 2 . 0 mg / kg CX5461 . ( n = 5 )

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Skin graft survival curve in mice treated with DMSO, 2.0 mg/kg FK506 or 0.5 -2.0 mg/kg CX5461. (n = 5)


Representative of HE of skin graft at seven days post transplantation . Scale bar , 50 μm .

Representative images of HE staining of skin graft at seven days post transplantation. Scale bar, 50 μm.


CD3 + T ( upper ) or CD4 + CD8 + T ( lower ) in spleens of skin - graft recipient mice were analyzed by FACS .

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CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of skin-graft recipient mice were analyzed by FACS.


G - H ELISA of IL - 2 IFN - γ in skin - graft recipient mouse serum at 4th day post transplantation . ( n = 5 )

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G-H ELISA analysis of IL-2 and IFN-γ levels in skin-graft recipient mouse serum at 4th day post transplantation. (n = 5)


survival time ( days ) of cardiac graft in mice treated with DMSO , 2 . 0 mg / kg FK506 or 2 . 0 mg / kg CX5461 . ( n = 5 )

The survival time (days) of cardiac graft in mice treated with DMSO, 2.0 mg/kg FK506 or 2.0 mg/kg CX5461. (n=5)


Representative of HE CD3 - of cardiac graft at seven days post transplantation . NC , Scale bar , 100 μm .

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Representative images of HE and CD3-IHC staining of cardiac graft at seven days post transplantation. NC, Scale bar, 100 μm.


CD3 + T ( upper ) or CD4 + CD8 + T ( lower ) in spleens of heart - graft recipient mice were analyzed by FACS .

CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of heart-graft recipient mice were analyzed by FACS.


L - M ELISA of IL - 2 IFN - γ in heart - graft recipient mouse serum at 7th day post transplantation . ( n = 5 )

L-M ELISA analysis of IL-2 and IFN-γ levels in heart-graft recipient mouse serum at 7th day post transplantation. (n = 5)


qPCR of pre - rRNA expression from transplanted mouse spleen . ( n = 5 )

qPCR analysis of pre-rRNA expression from transplanted mouse spleen. (n = 5)


FACS of CD4 + , CD25 + Foxp3 + Treg isolated from superficial cervical lymph nodes of heart - transplanted mice .

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FACS analysis of CD4+, CD25+ and Foxp3+ Treg cells isolated from superficial cervical lymph nodes of heart-transplanted mice.


B : Growth charts of 2 probands each showing typical growth patterns of self - limited DP , without compromised linear growth before puberty . Solid horizontal black lines connect green dots representing bone to red dots at equivalent chronological .

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B: Growth charts of 2 probands each showing typical growth patterns of self-limited DP, without compromised linear growth before puberty. Solid horizontal black lines connect green dots representing bone age to red dots at the equivalent chronological age.


C : Biological consequences of 2 identified N - terminal mutations . Both WT mutant N - terminal fragments ( p . Arg156Leu p . Glu161Lys ) were expressed in HEK293 as demonstrated by western blotting . GFP - tagged products of both were not detected in conditioned media of mammalian , as compared to wild type ( WT ) , appear to be retained in intracellular compartment ( mean ± s . e . m . ; n = 3 ) . Ponceau redstaining is shown to demonstrate equal loading for conditioned media . UT - untransfected negative control ; nd - not detected ; * : p = 0 . 01094 ( WT vs p . Arg156Leu ) p = 0 . 04408 ( WT vs p . Glu161Lys ) by 2 - t - . It has to date not been possible to retention for two C - terminal mutations due to difficulty expressing full or C - terminal fragment in mammalian .

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C: Biological consequences of the 2 identified N-terminal mutations. Both WT and mutant N-terminal protein fragments (p.Arg156Leu and p.Glu161Lys) were expressed in HEK293 cells as demonstrated by western blotting. The GFP-tagged protein products of both were not detected in the conditioned media of mammalian cells, as compared to wild type (WT), and appear to be retained in the intracellular compartment (mean ± s.e.m.; n = 3). Ponceau redstaining is shown to demonstrate equal protein loading for conditioned media. UT - untransfected negative control; nd - not detected; *: p=0.01094 (WT vs p.Arg156Leu) and p=0.04408 (WT vs p.Glu161Lys) by 2-tailed t-test. It has to date not been possible to test cytoplasmic retention for the two C-terminal mutations due to difficulty expressing the full length or C-terminal protein fragment in mammalian cells.


A - J : Igsf10 expression was not observed at E10 . 5 ( A , sagittal section ) but readily detectable at E12 . 5 , in NM ( B C , frontal sections ) E14 . 5 , along migratory path of GnRH neurons ( D , frontal section ) . Igsf10 positive were negative for Isolectin b4 ( marker of vasculature ) at developmental stages ( E , at E14 . 5 ) . Incubation with sense probe resulted in no signal at stages ( E14 . 5 shown in F ) . Peripherin - positive olfactory axons were extending within Igsf10 - positive NM ( G H ) . Cxcl12 mRNA expression is shown in I ( E14 . 5 , frontal section ) . GnRH neurons are located in MPOA by E17 . 5 ( J , frontal section ) in an Igsf10 negative .

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A-J: Igsf10 expression was not observed at E10.5 (A, sagittal section) but readily detectable at E12.5, in the NM (B and C, frontal sections) and E14.5, along the migratory path of GnRH neurons (D, frontal section). Igsf10 positive cells were negative for Isolectin b4 (marker of vasculature) at all developmental stages (E, at E14.5). Incubation with the sense probe resulted in no signal at all stages (E14.5 shown in F). Peripherin-positive cells olfactory axons were extending within the Igsf10-positive NM (G and H). Cxcl12 mRNA expression is shown in I (E14.5, frontal section). GnRH neurons are located in the MPOA by E17.5 (J, frontal section) in an Igsf10 negative area.


K - N : In human 11pcw brains , IGSF10 expression pattern was similar to that observed in mouse , with GnRH neurons interspersed in an IGSF10 - positive NM ( K - N , frontal sections , see also schematic in P ) . IGSF10 positive were also detected in olfactory bulb ( OB , panel O ) . Sense probe resulted in no specific signal ( panel N ) .

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K-N: In human 11pcw brains, IGSF10 expression pattern was similar to that observed in mouse, with GnRH neurons interspersed in an IGSF10-positive NM (K-N, frontal sections, see also schematic in P). IGSF10 positive cells were also detected in the olfactory bulb (OB, panel O). Sense probe resulted in no specific signal (panel N).


A : of Igsf10 expression in native NIH3T3 , stably expressing a scrambled ( sh - scr ) or Igsf10 ( sh - Igsf10 ) shRNA .

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A: Levels of Igsf10 expression in native NIH3T3 cells, and cells stably expressing a scrambled (sh-scr) or Igsf10 (sh-Igsf10) shRNA.


B C : migration of GN11 from into NIH3T3 sh - scr ( B ) or sh - Igsf10 ( C ) . D : of migration index ( see Methods ) .

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B and C: migration of GN11 cells from aggregates into NIH3T3 sh-scr (B) or sh-Igsf10 (C).D: Analysis of the migration index (see Methods).


E : RT - PCR in zebrafish of total larvae showing expression of Igsf10 mRNA at different time points . Expected band : 905 bp .

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E: RT-PCR analysis in zebrafish of total embryos and larvae showing the expression of Igsf10 mRNA at different time points. Expected band size: 905 bp.


F : Representative time - lapse of mispair control ( Control Sp - MO Control ATG - MO ) Igsf10 morpholinos ( Igsf10 Sp - MO Igsf10 ATG - MO ) injected Tg ( gnrh3 : EGFP ) larvae , at different time - points . show lateral view of live larvaehead ( 48 72 hpf ) dorsal view ( 4 5 dpf ) of live larvaehead ; anterior is left . arrowheads indicate normal GnRH3 neuron clusters in olfactory of control . Brackets show extension of projections towards hypothalamus . arrows indicate examples of abnormal GnRH3 neurons scattered in olfactory of morphants accompanied by lack of projections toward hypothalamus , indicated with Δ ( hpf - hours post fertilisation ; dpf - days post fertilisation . ) Scale bar , 100 um ( panels at 48 72hpf in F ) , 150 um ( panels at 4 5dpf in F ) . Panel G : Effect of Igsf10 Splice - site morpholino injection observed at 48hrs . of of showing an abnormal GnRH3 - neuron phenotype was significantly higher in Igsf10 Splice - site - MO ( Sp - MO ) injected compared to relative controls . * : p = 0 . 02422 , * * : p = 0 . 00101 , * * * : p = 0 . 00020 , * * * * : p = 0 . 00001 .

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F: Representative time-lapse analysis of mispair control (Control Sp-MO and Control ATG-MO) and Igsf10 morpholinos (Igsf10 Sp-MO and Igsf10 ATG-MO) injected Tg(gnrh3:EGFP) embryos and larvae, at different time-points. Images show lateral view of live larvaehead (48 and 72 hpf) and dorsal view (4 and 5 dpf) of live larvaehead; anterior is left. White arrowheads indicate normal GnRH3 neuron clusters in the olfactory area of control embryos. Brackets show the extension of the projections towards the hypothalamus. White arrows indicate examples of abnormal GnRH3 neurons scattered in the olfactory area of morphants accompanied by lack of projections toward the hypothalamus, indicated with Δ (hpf - hours post fertilisation; dpf - days post fertilisation.) Scale bar, 100 um (panels at 48 and 72hpf in F), 150 um (panels at 4 and 5dpf in F). Panel G: Effect of Igsf10 Splice-site morpholino injection observed at 48hrs. Quantification of the percentage of embryos showing an abnormal GnRH3-neuron phenotype was significantly higher in Igsf10 Splice-site-MO (Sp-MO) injected embryos compared to relative controls. *: p=0.02422, **: p=0.00101, ***: p=0.00020, ****: p=0.00001.


( A ) 12 - lead ECG of 1 . ( B ) 12 - lead ECG of 2 . ( C ) ECG of 2 during epinephrine ( A - represents baseline ECG ; B - ECG at 17 min 47 s of beginning of epinephrine infusion ( 0 . 2 μg / kg of epinephrine ) C - ECG after end of epinephrine infusion ) .

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(A) 12-lead ECG of patient 1. (B) 12-lead ECG of patient 2. (C) ECG of patient 2 during epinephrine test (A- represents baseline ECG; B- ECG at 17 min and 47 s of the beginning of epinephrine infusion (0.2 μg/kg of epinephrine) and C- ECG after the end of epinephrine infusion).


( E ) ECG of 3 ( subject IV : 13 ) showing mild QTc prolongation .

(E) ECG of patient 3 (subject IV:13) showing mild QTc prolongation.


( F ) ICD interrogation of 3 ( subject IV : 13 ) reveals an episode of VT .

(F) ICD interrogation of patient 3 (subject IV:13) reveals an episode of VT.


( A - H ) Expression of Tecrl in mouse development . Expression is not observed in ( A ) cardiac crescent at E7 . 5 but is prominent in ( B ) inflow tract ( ift ) region at E8 . 5 , particularly in ( C ) left sinus venosus ( lsv ) . ( D ) At E9 . 5 , Tecrl expression is observed in four chambers of heart but is strongest in inflow tract . ( E - G ) From E10 onwards , Tecrl is expressed in somites . ( H ) At E14 . 5 , Tecrl expression is present in entire myocardium ; [ atrium ( a ) , ventricle ( v ) , neural tube ( nt ) , lung ( lu ) , right atrium ( ra ) , left atrium ( la ) , right ventricle ( rv ) left ventricle ( lv ) ] .

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(A-H) Expression of Tecrl in mouse development. Expression is not observed in (A) the cardiac crescent at E7.5 but is prominent in (B) the inflow tract (ift) region at E8.5, particularly in (C) the left sinus venosus (lsv). (D) At E9.5, Tecrl expression is observed in all four chambers of the heart but is strongest in the inflow tract. (E-G) From E10 onwards, Tecrl is expressed in the somites. (H) At E14.5, Tecrl expression is present in the entire myocardium; [atrium (a), ventricle (v), neural tube (nt), lung (lu), right atrium (ra), left atrium (la), right ventricle (rv) and left ventricle (lv)].


( I ) mRNA expression analyses of TECR TECRL by RT - qPCR in human tissues demonstrates preferential expression of Tecrl in heart ; [ Bone marrow ( Bone M . ) , heart , skeletal muscle ( Sk . Muscle ) , uterus , liver , spleen , thymus , thyroid , prostate , brain , lung , small intestine ( Small I . ) colon ]

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(I) mRNA expression analyses of TECR and TECRL by RT-qPCR in human tissues demonstrates preferential expression of Tecrl in the heart; [Bone marrow (Bone M.), heart, skeletal muscle (Sk.Muscle), uterus, liver, spleen, thymus, thyroid, prostate, brain, lung, small intestine (Small I.) and colon]


( J ) Localization of MYC - Tecrl in COS - 1 . accumulation of MYC - Tecrl is perinuclear consistent with it being localized to ER .

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(J) Localization of MYC-Tecrl in COS-1 cells. The accumulation of MYC-Tecrl is perinuclear consistent with it being localized to the ER.


( K - M ) Co - localization of MYC - Tecrl ER marker Calnexin in H10 . Nuclei stained with DAPI . Scale bar : 10 μm .

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(K-M) Co-localization of MYC-Tecrl and the ER marker Calnexin in H10 cells. Nuclei stained with DAPI. Scale bar: 10 μm.


( A ) Skin fibroblasts ( left ) from IV : 13 , homozygous for TECRLc . 331 + 1G > A mutation were reprogrammed to hiPSCs ( center ) , which expressed pluripotency markers NANOG SSEA4 ( right ) . Scale bars : 100 μm .

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(A) Skin fibroblasts(left) from IV:13, homozygous for the TECRLc.331+1G>A mutation were reprogrammed to hiPSCs(center), which expressed the pluripotency markers NANOG and SSEA4(right). Scale bars: 100 μm.


( B ) TECRLHom - hiPSCs have a normal karyotype ( left ) PluriTest demonstrates a high pluripotency score low novelty score for three hiPSC lines ( right ) .

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(B) TECRLHom-hiPSCs have a normal karyotype(left) and PluriTest demonstrates a high pluripotency score and low novelty score for all three hiPSC lines (right).


( C ) TECRLHom - hiPSCs generate derivatives of mesoderm ( left ) , endoderm ( center ) ectoderm ( right ) . Scale bars : 25

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(C) TECRLHom-hiPSCs generate derivatives of mesoderm(left), endoderm(center) and ectoderm(right). Scale bars: 25 μm


( F ) ACTN2 immunostaining of CTRL - , TECRLHet - TECRLHom - hiPSC - CMs . Scale bar : 25 μm .

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(F) ACTN2 immunostaining of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs. Scale bar: 25 μm.


( G ) of RT - PCR products by gel electrophoresis . Amplification products of TECRL exons 2 - 4 from CTRL - , TECRLHet - TECRLHom - hiPSC - CMs ( left ) coding of TECRL from CTRL - TECRLHom - hiPSC - CMs ( right ) .

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(G) Analysis of RT-PCR products by gel electrophoresis. Amplification products of TECRL exons 2-4 from CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs(left) and coding sequence of TECRL from CTRL- and TECRLHom-hiPSC-CMs(right).


( A ) Representative traces of [ Ca2 + ] i transients in Indo - 1 AM - loaded hiPSC - CMs paced at 1 Hz . ( B ) Time to reach 50 % of [ Ca2 + ] i transient amplitude , t1 / 2 ; [ Ca2 + ] i concentrations during systole diastole ( C ) Amplitude tau decay of [ Ca2 + ] i transient .

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(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz. (B) Time to reach 50% of [Ca2+]i transient amplitude, t1/2; [Ca2+]i concentrations during systole and diastole (C) Amplitude and tau decay of the [Ca2+]i transient.


( A ) Representative traces of [ Ca2 + ] i transients in Indo - 1 AM - loaded hiPSC - CMs paced at 1 Hz , in presence of caffeine ( caff ) or caffeine NiCl2 . ( B ) Amplitude of [ Ca2 + ] i in presence of caffeine NiCl2and fractional SR release in hiPSC - CMs .

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(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz, in the presence of caffeine (caff) or caffeine and NiCl2. (B) Amplitude of [Ca2+]i in the presence of caffeine and NiCl2and fractional SR release in hiPSC-CMs.


( C ) Rate constants of SERCA - , NCX - slow mechanisms - [ Ca2 + ] i decay in hiPSC - CMs . ( D ) Relative contribution of SERCA , NCX slow mechanisms to [ Ca2 + ] i extrusion in hiPSC - CMs .

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(C) Rate constants of SERCA-, NCX- and slow mechanisms-based [Ca2+]i decay in hiPSC-CMs. (D) Relative contribution of SERCA, NCX and slow mechanisms to [Ca2+]i extrusion in hiPSC-CMs.


( A ) AP illustrating analysed parameters ( B ) Representative APs from control ( CTRL ) , heterozygous ( HET ) homozygous ( HOM ) CMs . ( C ) RMP , dV / dtmax , APAmax , APAplat , APD20 , APD50 APD90 of CTRL - , TECRLHet - TECRLHom - hiPSC - CMs .

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(A) AP illustrating the analysed parameters (B) Representative APs from control (CTRL), heterozygous (HET) and homozygous (HOM) CMs. (C) RMP, dV/dtmax , APAmax, APAplat, APD20, APD50 and APD90 of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs.


( A ) Representative AP traces ( B ) averaged activity of triggered spontaneous activity in hiPSC - CMs in absence of NA . ( C ) Representative AP traces ( D ) averaged activity of triggered spontaneous activity in hiPSC - CMs in presence of NA .

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(A) Representative AP traces and (B) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the absence of NA. (C) Representative AP traces and (D) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the presence of NA.


( A ) Representative AP trace of a TECRLHom - hiPSC - CM in presence of NA alone or NA flecainide . ( B ) Effect of flecainide on AP parameters of hiPSC - CMs . ( C - D ) Addition of 5 μM of flecainide decreased susceptibility to triggered activity in hiPSC - CMs . Note that effect of flecainide on triggered activity of TECRLHet - TECRLHom - hiPSC - CMs is more pronounced than its effect on their spontaneous activity .

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(A) Representative AP trace of a TECRLHom-hiPSC-CM in the presence of NA alone or NA and flecainide. (B) Effect of flecainide on AP parameters of hiPSC-CMs. (C-D) Addition of 5 μM of flecainide decreased the susceptibility to triggered activity in hiPSC-CMs. Note that the effect of flecainide on triggered activity of TECRLHet- and TECRLHom-hiPSC-CMs is more pronounced than its effect on their spontaneous activity.


A , B Detection of γb palmitoylation through biotin - switch in N . benthamiana . γb - 3xFlag γb3CS - 3xFlag were treated with ( Hyd + ) or without ( Hyd - ) hydroxylamine , thioester cleavage reagent . Lanes labelled ' Palmitoylation ' show γb - 3xFlag or γb3CS - 3xFlag amounts recovered from neutravidin beads . loading controls indicate sample loading onto neutravidin beads . Data information : In ( A , representative data are shown three biological replicates had similar results .

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A, B Detection of γb palmitoylation through the biotin-switch assay in N. benthamiana. The γb-3xFlag and γb3CS-3xFlag proteins were treated with (Hyd+) or without (Hyd-) hydroxylamine, the thioester cleavage reagent. Lanes labelled ' Palmitoylation ' show γb-3xFlag or γb3CS-3xFlag amounts recovered from the neutravidin beads. The loading controls indicate sample loading onto the neutravidin beads. Data information: In (A, representative data are shown and three biological replicates had similar results.


D Western blot detection of viral accumulation in BSMV - BSMV3CS - infected N . benthamiana with anti - TGB1 or anti - γb antibodies . SL8 , eighth systemically infected leaf ; SL9 , ninth systemically infected leaf . Ino , agroinfiltrated leaves . Rubisco large subunit ( RbcL ) was used as a loading control . Data information : In representative data are shown three biological replicates had similar results .

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D Western blot detection of viral accumulation in BSMV- and BSMV3CS-infected N. benthamiana plants with anti-TGB1 or anti-γb antibodies. SL8, eighth systemically infected leaf; SL9, ninth systemically infected leaf. Ino, agroinfiltrated leaves. Rubisco large subunit (RbcL) was used as a loading control. Data information: In representative data are shown and three biological replicates had similar results.


F Western blot of accumulation in BSMV - BSMV3CS - systemically infected barley leaves with anti - TGB1 or anti - γb antibodies . RbcL was used as a loading control . Data information : In F ) , representative data are shown three biological replicates had similar results .

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F Western blot analysis of protein accumulation in BSMV- and BSMV3CS- systemically infected barley leaves with anti-TGB1 or anti-γb antibodies. RbcL was used as a loading control. Data information: In F), representative data are shown and three biological replicates had similar results.


A Effects of γb palmitoylation on BSMV replication . Movement - deficient BSMV ( RNAα + RNAγ / RNAγ3CS ) was agroinfiltrated into N . benthamiana leaves . At 3 dpi , total were extracted for Western Northern blot analyses . Antibodies probes used for molecular analyses are indicated on right of each panel . Actin was used to monitor equal loading , methylene blue - stained rRNAs served as loading controls . Data information : representative data are shown three biological replicates had similar results .

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A Effects of γb palmitoylation on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ/RNAγ3CS) was agroinfiltrated into N. benthamiana leaves. At 3 dpi, total protein and RNA were extracted for Western and Northern blot analyses. Antibodies and probes used for molecular analyses are indicated on the right of each panel. Actin was used to monitor equal protein loading, and methylene blue-stained rRNAs served as RNA loading controls. Data information: representative data are shown and three biological replicates had similar results.


B Western blot analyses of chloroplast localization of γb γb3CS . Transiently expressed αa - GFP in intact chloroplasts isolated from BSMVγb - 3xFlag - or BSMV3CS - 3xFlag - infected N . benthamiana leaves at 4 dpi . Detection of γb - 3xFlag γb3CS - 3xFlag with an anti - Flag antibody ; TGB1 with an anti - TGB1 antibody ; αa - GFP detection with an anti - GFP antibody . NTRC was a loading control for intact chloroplasts Phosphoenolpyruvate carboxylase ( PEPC ) was used to assess contamination of isolated chloroplasts . Data information : , representative data are shown three biological replicates had similar results .

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B Western blot analyses of chloroplast localization of γb and γb3CS. Transiently expressed αa-GFP in intact chloroplasts isolated from BSMVγb-3xFlag- or BSMV3CS-3xFlag-infected N. benthamiana leaves at 4 dpi. Detection of γb-3xFlag and γb3CS-3xFlag with an anti-Flag antibody; TGB1 analysis with an anti-TGB1 antibody; αa-GFP detection with an anti-GFP antibody. NTRC was a loading control for intact chloroplasts Phosphoenolpyruvate carboxylase (PEPC) was used to assess cytosolic contamination of isolated chloroplasts. Data information: , representative data are shown and three biological replicates had similar results.


C Subcellular localization of γb - GFP γb3CS - GFP in N . benthamiana epidermal leaf . first second rows show subcellular localization of γb - GFP γb3CS - GFP transient expression in leaves . third fourth rows show localization of γb - GFP γb3CS - GFP in BSMVγb - GFP - BSMV3CS - GFP - infected leaves at 3 dpi . Figures on right indicate normalized intensities of GFP ( green ) or chloroplast autofluorescence ( red ) channels along dashed lines shown in merged of left panel . Scale bars , 20 μm . Data information : representative data are shown three biological replicates had similar results .

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C Subcellular localization of γb-GFP and γb3CS-GFP in N. benthamiana epidermal leaf cells. The first and second rows show subcellular localization of γb-GFP and γb3CS-GFP transient expression in leaves. The third and fourth rows show localization of γb-GFP and γb3CS-GFP in BSMVγb-GFP- and BSMV3CS-GFP-infected leaves at 3 dpi. Figures on the right indicate the normalized fluorescence intensities of the GFP (green) or the chloroplast autofluorescence (red) channels along the dashed white lines shown in the merged images of the left panel. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.


D Subcellular localization of γb - GFP or γb3CS - GFP in isolated N . benthamiana protoplasts harvested at 3 dpi . fifth sixth columns show γb localization in BSMVγb - GFP - ( movement - deficient ) N . benthamiana protoplasts after treatment with 200 μM 2 - BP inhibitor . released protoplasts were collected after 48 h incubation in DMSO or 2 - BP followed by confocal microscopy visualization . Figures at bottom indicate normalized intensities of GFP ( green ) chloroplast autofluorescence ( red ) channels along dashed lines in merged above . Scale bars , 20 μm . Data information : representative data are shown three biological replicates had similar results .

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D Subcellular localization of γb-GFP or γb3CS-GFP in isolated N. benthamiana protoplasts harvested at 3 dpi. The fifth and sixth columns show γb localization in BSMVγb-GFP- (movement-deficient) N. benthamiana protoplasts after treatment with 200 μM 2-BP inhibitor. The released protoplasts were collected after 48 h incubation in DMSO or 2-BP followed by confocal microscopy visualization. Figures at the bottom indicate the normalized fluorescence intensities of the GFP (green) and the chloroplast autofluorescence (red) channels along the dashed white lines in the merged images above. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.


A Effects of γb palmitoylation on - to - movement at 3 dpi with BSMV duplex ( dfBSMV ) reporter system dfBSMVmγb mutant control contains an ATG substitution of γb initiation codon Red produced from mCherry expression cassette illustrates infection foci , whereas GFP outside red region shows invaded surrounding infection foci . was evaluated by confocal microscopy . Scale bars , 100 μm . B of movement in Panel A . To determine movement capacity of depicted in each panel , mCherry GFP intensities were first determined by ImageJ software . Then , intensities within secondarily invaded were divided by mCherry plus GFP intensities in infection foci ( n = 10 ) . Y - axis indicates relative sizes of green areas in comparison to that of red - colored areas . Data information : In ( A , representative data are shown three biological replicates had similar results . In ( B ) , box boundaries indicate upper ( 25th percentile ) lower ( 75th percentile ) quartiles , whiskers indicate minimum maximum values , central band indicates median . Letters in chart denote statistically significant differences among groups according to Duncan ' s multiple range ( p < 0 . 05 ) .

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A Effects of γb palmitoylation on virus cell-to-cell movement at 3 dpi with the BSMV duplex fluorescence (dfBSMV) reporter system The dfBSMVmγb mutant control contains an ATG substitution of the γb initiation codon Red fluorescence produced from the mCherry expression cassette illustrates the primary infection foci, whereas GFP fluorescence outside the red region shows invaded cells surrounding the primary infection foci. Fluorescence was evaluated by confocal microscopy. Scale bars, 100 μm. B Quantification of virus movement in Panel A. To determine the movement capacity of the viruses depicted in each panel, the mCherry and GFP fluorescent intensities were first determined by ImageJ software. Then, the intensities within the secondarily invaded cells were divided by the fluorescent mCherry plus the GFP intensities in the primary infection foci (n = 10). The Y-axis indicates the relative sizes of the green areas in comparison to that of the red-colored areas. Data information: In (A, representative data are shown and three biological replicates had similar results. In (B), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


D Biotin - switch to access palmitoylation of γb in BSMV - infected N . benthamiana leaves at different timepoints . BSMVγb - 3xFlag was treated with hydroxylamine ( Hyd + ) thioester cleavage reagent . Equal sample amounts used for palmitoylation analyses were monitored by Western blot with an anti - Flag antibody . Data information : In representative data are shown three biological replicates had similar results .

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D Biotin-switch assay to access palmitoylation of γb in BSMV-infected N. benthamiana leaves at different timepoints. BSMVγb-3xFlag was treated with the hydroxylamine (Hyd+) thioester cleavage reagent. Equal sample amounts used for palmitoylation analyses were monitored by Western blot analysis with an anti-Flag antibody. Data information: In representative data are shown and three biological replicates had similar results.