Figure 7 ( A ) HIV - 1 - infected primary CD4 T cells ( strain CH058 ) were incubated with sera of HIV negative ( HIV - ; n = 9 ) or ART - treated HIV - 1 - infected ( HIV + ; n = 9 ) individuals ( serum dilution 1 : 1000 ) . The levels of IgG binding on infected ( Gag + ) cells were analyzed by flow cytometry . Results are expressed as the MFI on infected ( Gag + ) cells . Error bars indicate SEM . * * * * P < 0 , 0001 ; Mann - Whitney test . ( B ) CH058 - infected primary CD4 T cells were cultured for 24h with sera of healthy ( HIV - ; n = 9 ) or ART - treated HIV - 1 - infected ( HIV + ; n = 9 ) individuals ( serum dilution 1 : 100 ) and 50 % NHS from a healthy donor . C3 surface levels on infected ( Gag + ) cells were analyzed by flow cytometry . Results are expressed as the MFI on infected ( Gag + ) cells . Error bars indicate SEM . * * * * P < 0 , 0001 ; Mann - Whitney test . ( C ) CD4 T cells not infected ( NI ) or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( strain CH058 ) were incubated with the serum of HIV - 1 - infected ART - treated individuals ( sKB104 or sKB105 , dilution 1 : 1000 ) . The levels of IgG binding on infected ( Gag + ) cells were analyzed by flow cytometry . One representative experiment is shown on the left . On the right , results are expressed as MFI of staining . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells . Error bars indicate SEM . * P < 0 . 05 , Wilcoxon test . Only significant comparisons are depicted . ( D ) Primary CD4 T cells either not infected ( NI ) or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( strain CH058 ) were incubated with 50 % NHS from a healthy donor and serum of HIV - 1 - infected ART - treated individuals ( sKB104 or sKB105 , dilution 1 : 100 ) . The levels of C3 deposition on infected ( Gag + ) cells were analyzed by flow cytometry . One representative experiment is shown ( left panel ) . Results are expressed as MFI of staining ( right panel ) . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells . ( E ) Correlation between patients ' sera ( sK104 and sKB105 ) antibody binding and C3 deposition on CD4 T cells either mock - infected or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( strain CH058 ) . Each dot is the mean of 6 donors of CD4 T cells . Correlation was analyzed by Spearman correlation . Correlation coefficient ( r ) and p - value are indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) HIV-1-infected primary CD4 T cells (strain CH058) were incubated with sera of HIV negative (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.(B) CH058-infected primary CD4 T cells were cultured for 24h with sera of healthy (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:100) and 50% NHS from a healthy donor. C3 surface levels on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.(C) CD4 T cells not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with the serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown on the left. On the right, results are expressed as MFI of staining. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Error bars indicate SEM. *P<0.05, Wilcoxon test. Only significant comparisons are depicted.(D) Primary CD4 T cells either not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with 50% NHS from a healthy donor and serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:100). The levels of C3 deposition on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown (left panel). Results are expressed as MFI of staining (right panel). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.(E) Correlation between patients' sera (sK104 and sKB105) antibody binding and C3 deposition on CD4 T cells either mock-infected or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058). Each dot is the mean of 6 donors of CD4 T cells. Correlation was analyzed by Spearman correlation. Correlation coefficient (r) and p-value are indicated.


Figure 1A T98G cells were challenged or not with dsDNAs or with 2 ' 3 ' cGAMP for 6 h prior to whole cell extraction and Western Blot ( WB ) analysis using indicated antibodies . C Gli4 cells were treated as in A prior to WB analysis using indicated antibodies . 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 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 cell extracts from T98G cells treated or not for 72 h with 0 . 16 µM CPT were analyzed by WB using indicated antibodies .

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Figure 1A T98G cells were challenged or not with dsDNAs or with 2'3'cGAMP for 6 h prior to whole cell extraction and Western Blot (WB) analysis using indicated antibodies.C Gli4 cells were treated as in A prior to WB analysis using indicated antibodies.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 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 cell extracts from T98G cells treated or not for 72 h with 0.16 µM CPT were analyzed by WB using indicated antibodies.


Figure 2A 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 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 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 .

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Figure 2A 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 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 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.


Figure 3A 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 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 analysis using indicated antibodies . K THP - 1 were transfected or not with dsDNA in presence or not of NU7441 , prior to WB analysis using indicated antibodies .

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Figure 3A 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 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 analysis using indicated antibodies.K THP-1 were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.


Figure 4F 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 ) .

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Figure 4F 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).


Figure 5A 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 the quantification of macrophages recruited at tumor site 24 h post xenotransplantation in D ( n = 21 T98G - GFPEmpty and n = 29 T98G - GFPcGAS embryos ) .

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Figure 5A 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 the quantification of macrophages recruited at tumor site 24 h post xenotransplantation in D (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).


Figure 6G Graph presents the survival rate of glioblastoma patients from A that present PRKDClow / MB21D1low , PRKDChigh / MB21D1low and PRKDChigh / MB21D1high expression . H Violin plots present the expression of PRKDC and MB21D1 in datasets analyzed in A , based on tumor grade ( II to IV ) .

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Figure 6G Graph presents the survival rate of glioblastoma patients from A that present PRKDClow/MB21D1low, PRKDChigh/MB21D1low and PRKDChigh/MB21D1high expression.H Violin plots present the expression of PRKDC and MB21D1 in datasets analyzed in A, based on tumor grade (II to IV).


Figure 13D - 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 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 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 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 ( white arrows ) in presence of endogenous mouse tau in ECrTgTau mice . Scale bars 50 µm . 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 ) 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 ) .

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Figure 13D-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 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 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 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 (white arrows) in presence of endogenous mouse tau in ECrTgTau mice. Scale bars 50 µm.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)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).


Figure 2Primary 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 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 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 .

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Figure 2Primary 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 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 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.


Figure 3A . 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 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 .

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Figure 3A. 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 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.


Figure 4Human , 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 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 ± SEM , n = 3 mice / group . 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 . 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 ) 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 .

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Figure 4Human, 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 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±SEM, n=3 mice/group.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.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) 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.


Figure 5Cortical 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 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 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 ) 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 ) 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 .

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Figure 5Cortical 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 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 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) 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) 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.


Figure 6Extraction 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 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 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 .

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Figure 6Extraction 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 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 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.


Figure 2A - 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 . )

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Figure 2A-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.)


Figure 3LSM 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 µmLSM 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 ) ,

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Figure 3LSM 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 µmLSM 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),


Figure 4Bright - 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 LECLSM 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 µmNumber of LECs per jls / pTD section in E13 . 5 control or ILK & β1 integrin K . O . embryosLEC 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 . embryosQuantification of the 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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Figure 4Bright-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 LECLSM 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 µmNumber of LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryosLEC 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. embryosQuantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK & β1 integrin K.O. embryos


Figure 5LSM 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 , respectivelyDermal 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 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 ) 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 ) ,

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5LSM 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, respectivelyDermal 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 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)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),


Figure 6LSM 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 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 . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6LSM 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 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.)


Figure 7Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA . Scale bars : 50 µmLEC 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 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 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 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 µmLEC 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 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 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)


Figure 8LSM 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 , 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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8LSM 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, 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


Figure 1 Confocal images of HeLa cells stained with NF45 / NF90 antibodies and antibodies against nucleolin and fibrillarin . Scale bar , 10 μm . 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 . 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 )

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

Figure 1 Confocal images of HeLa cells stained with NF45/NF90 antibodies and antibodies against nucleolin and fibrillarin. Scale bar, 10 μm.  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.    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)


Figure 2 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 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 . 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 .

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

Figure 2  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 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.  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.


Figure 3 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 analysis of endogenous UBF with NF45 and NF90 in HEK293 cells . 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 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 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 images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 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 analysis of endogenous UBF with NF45 and NF90 in HEK293 cells.  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 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 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 images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm.


Figure 4 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 analysis of pre - rRNA in CD3 + T cells treated with P / I for 6 h . ( n = 3 ) 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 ) The analysis of rDNA - luc activity in Jurkat cells with or without NF90 silence upon P / I stimulation or not . ( n = 3 ) 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 analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P / I . ChIP analysis of NFATc2 binding to the rDNA loci in CD3 + T cells stimulated with P / I for 6 h . ( n = 3 ) 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 analysis of NFATc2 binding to the H42 . 9 loci in NF90 - slienced CD3 + T cells stimulated with P / I for 6 h . ( n = 3 )

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

Figure 4 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 analysis of pre-rRNA in CD3+ T cells treated with P/I for 6 h. (n=3)  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)  The analysis of rDNA-luc activity in Jurkat cells with or without NF90 silence upon P/I stimulation or not. (n=3)  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 analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P/I.  ChIP analysis of NFATc2 binding to the rDNA loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)  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 analysis of NFATc2 binding to the H42.9 loci in NF90-slienced CD3+ T cells stimulated with P/I for 6 h. (n=3)


Figure 5 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 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 ) The proliferation of shNC , shNF45 or shNF90 - infected PBMCs was monitored using a CCK - 8 assay . ( n = 3 ) 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 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 and IFN - γ in CD3 + T cells with the indicated treatments . ( n = 3 ) Flow cytometry analysis of CFSE - labeled CD3 + T cells in the presence of DMSO , FK506 and CX5461 for 0 , 3 and 7 d .

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

Figure 5 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 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)  The proliferation of shNC, shNF45 or shNF90-infected PBMCs was monitored using a CCK-8 assay. (n = 3)  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 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 and IFN-γ in CD3+ T cells with the indicated treatments. (n = 3)  Flow cytometry analysis of CFSE-labeled CD3+ T cells in the presence of DMSO, FK506 and CX5461 for 0, 3 and 7 d.


Figure 6 qPCR analysis of pre - rRNA expression from skin - and heart - transplanted mouse spleens . ( n = 5 ) 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 ) Representative images of HE staining of skin graft at seven days post transplantation . Scale bar , 50 μm . 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 analysis of IL - 2 and IFN - γ levels in skin - graft recipient mouse serum at 4th day post transplantation . ( 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 images of HE and CD3 - IHC staining of cardiac graft at seven days post transplantation . NC , Scale bar , 100 μm . 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 analysis of IL - 2 and IFN - γ levels in heart - graft recipient mouse serum at 7th day post transplantation . ( n = 5 ) qPCR analysis of pre - rRNA expression from transplanted mouse spleen . ( n = 5 ) FACS analysis of CD4 + , CD25 + and Foxp3 + Treg cells isolated from superficial cervical lymph nodes of heart - transplanted mice .

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

Figure 6 qPCR analysis of pre-rRNA expression from skin- and heart-transplanted mouse spleens. (n = 5)  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)  Representative images of HE staining of skin graft at seven days post transplantation. Scale bar, 50 μm.  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 analysis of IL-2 and IFN-γ levels in skin-graft recipient mouse serum at 4th day post transplantation. (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 images of HE and CD3-IHC staining of cardiac graft at seven days post transplantation. NC, Scale bar, 100 μm.  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 analysis of IL-2 and IFN-γ levels in heart-graft recipient mouse serum at 7th day post transplantation. (n = 5)  qPCR analysis of pre-rRNA expression from transplanted mouse spleen. (n = 5)  FACS analysis of CD4+, CD25+ and Foxp3+ Treg cells isolated from superficial cervical lymph nodes of heart-transplanted mice.


Figure 2B : 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 .

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Figure 2B: 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.


Figure 3C : 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 .

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Figure 3C: 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.


Figure 4A - 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 cells were also detected in the olfactory bulb ( OB , panel O ) . Sense probe resulted in no specific signal ( panel N ) .

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Figure 4A-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 cells were also detected in the olfactory bulb (OB, panel O). Sense probe resulted in no specific signal (panel N).


Figure 5A : Levels of Igsf10 expression in native NIH3T3 cells , and cells stably expressing a scrambled ( sh - scr ) or Igsf10 ( sh - Igsf10 ) shRNA . 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 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 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 .

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Figure 5A: Levels of Igsf10 expression in native NIH3T3 cells, and cells stably expressing a scrambled (sh-scr) or Igsf10 (sh-Igsf10) shRNA.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 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 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.


Figure 1 ( 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 patient 3 ( subject IV : 13 ) showing mild QTc prolongation . ( F ) ICD interrogation of patient 3 ( subject IV : 13 ) reveals an episode of VT .

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Figure 1(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 patient 3 (subject IV:13) showing mild QTc prolongation.(F) ICD interrogation of patient 3 (subject IV:13) reveals an episode of VT.


Figure 2 ( 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 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 cells . The accumulation of MYC - Tecrl is perinuclear consistent with it being localized to the ER . ( K - M ) Co - localization of MYC - Tecrl and the ER marker Calnexin in H10 cells . Nuclei stained with DAPI . Scale bar : 10 μm .

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Figure 2(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 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 cells. The accumulation of MYC-Tecrl is perinuclear consistent with it being localized to the ER.(K-M) Co-localization of MYC-Tecrl and the ER marker Calnexin in H10 cells. Nuclei stained with DAPI. Scale bar: 10 μm.


Figure 3 ( 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 ) 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 ) and ectoderm ( right ) . Scale bars : 25 μm ( F ) ACTN2 immunostaining of CTRL - , TECRLHet - and TECRLHom - hiPSC - CMs . Scale bar : 25 μm . ( 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 ) .

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Figure 3(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) 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) and ectoderm(right). Scale bars: 25 μm(F) ACTN2 immunostaining of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs. Scale bar: 25 μm.(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).


Figure 4 ( 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 .

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Figure 4(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.


Figure 5 ( 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 - 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 .

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Figure 5(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- 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.


Figure 6 ( 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 .

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Figure 6(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.


Figure 7 ( 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 .

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Figure 7(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.


Figure 8 ( 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 .

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Figure 8(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.


Figure 1A , 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 - 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, 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- 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 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.