Quantification of the percentage of cells exhibiting Golgi - nucleating activity in centrinone - treated cells 48h after the washout of the drug . Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63 . At least 100 cells from two independent experiments were analyzed in each condition .

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Quantification of the percentage of cells exhibiting Golgi-nucleating activity in centrinone-treated cells 48h after the washout of the drug. Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63. At least 100 cells from two independent experiments were analyzed in each condition.


Control ( top ) and centrinone - treated ( bottom ) RPE - 1 cells double stained for either AKAP450 , CDK5Rap2 , PCNT or CEP192 ( all shown in green ) and giantin ( red ) as a Golgi marker . DNA was counterstained with DAPI and is shown in blue . Single channel ( left , green ) and merged images ( right ) are shown in each case . White arrows indicate the centrosome ( CTR ) .

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Control (top) and centrinone-treated (bottom) RPE-1 cells double stained for either AKAP450, CDK5Rap2, PCNT or CEP192 (all shown in green) and giantin (red) as a Golgi marker. DNA was counterstained with DAPI and is shown in blue. Single channel (left, green) and merged images (right) are shown in each case. White arrows indicate the centrosome (CTR).


IF images of control and centrinone - treated RPE - 1 cells treated with NZ to induce GA fragmentation and double stained with either anti - AKAP450 , anti - CDK5Rap2 or anti - PCNT ( all shown in green ) and anti - giantin antibodies ( white ) . Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker . The yellow dashed line indicates the contour of the nucleus . Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces .

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IF images of control and centrinone-treated RPE-1 cells treated with NZ to induce GA fragmentation and double stained with either anti-AKAP450, anti-CDK5Rap2 or anti-PCNT (all shown in green) and anti-giantin antibodies (white). Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker. The yellow dashed line indicates the contour of the nucleus. Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces.


Box - and - whisker plots showing quantification of association between the indicated protein and Golgi elements Top and bottom ends of the boxes represent 75th and 25th percentiles , and whiskers represent 90th and 10th percentiles . The median is depicted with a solid line . Individual Golgi elements from at least 14 cells were delineated ( > 500 elements / experiment ) and the intensity of each protein spot associated with each Golgi element was measured . Data were collected from two independent experiments and normalized to WT mean .

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Box-and-whisker plots showing quantification of association between the indicated protein and Golgi elements Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Individual Golgi elements from at least 14 cells were delineated (>500 elements/experiment) and the intensity of each protein spot associated with each Golgi element was measured. Data were collected from two independent experiments and normalized to WT mean.


MT - regrowth experiment after NZ treatment in centrinone - treated cells . High magnification images at the bottom are representative of a triple staining with α - tubulin ( green ) , GM130 ( blue ) and the indicated protein ( red ) and show MTs growing from the surface of the GA . Double labelling without α - tubulin are also shown ( top ) to allow better visualization of PCM - protein spots on Golgi elements acting as MT nucleation sites ( arrowheads ) .

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MT-regrowth experiment after NZ treatment in centrinone-treated cells. High magnification images at the bottom are representative of a triple staining with α-tubulin (green), GM130 (blue) and the indicated protein (red) and show MTs growing from the surface of the GA. Double labelling without α-tubulin are also shown (top) to allow better visualization of PCM-protein spots on Golgi elements acting as MT nucleation sites (arrowheads).


Centrinone - treated pcnt KO , c5rap2 KO and akap KO cells , were incubated with NZ for 3h , fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated ( n = 3 ) .

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Centrinone-treated pcnt KO, c5rap2 KO and akap KO cells, were incubated with NZ for 3h, fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated (n=3).


MT regrowth assay in centrinone - treated WT , pcnt KO and akap KO RPE - 1 cells stained with antibodies to EB1 and giantin . At right , quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone , as a measure of MT - nucleation from the GA . Data were collected from two independent experiments and normalized to WT mean .

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MT regrowth assay in centrinone-treated WT, pcnt KO and akap KO RPE-1 cells stained with antibodies to EB1 and giantin. At right, quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone, as a measure of MT-nucleation from the GA. Data were collected from two independent experiments and normalized to WT mean.


Representative MT network of a centrosome - less akap KO cell .

Representative MT network of a centrosome-less akap KO cell.


MT - regrowth experiment after NZ treatment in centrinone - treated akap KO cells . A representative confocal image of a double labelling for EB1 ( green ) and giantin ( blue ) is shown . High magnification at right shows that growing MT asters are not associated to Golgi fragments .

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MT-regrowth experiment after NZ treatment in centrinone-treated akap KO cells. A representative confocal image of a double labelling for EB1 (green) and giantin (blue) is shown. High magnification at right shows that growing MT asters are not associated to Golgi fragments.


Centrinone - treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies . High magnification images of the boxed areas are shown as merge ( top ) or individual green or red labelling ( middle and bottom panels ) .

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Centrinone-treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies. High magnification images of the boxed areas are shown as merge (top) or individual green or red labelling (middle and bottom panels).


Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 ( top panels ) or PCNT ( bottom panels ) . After 3 - min MT regrowth , cells were fixed and stained with the indicated antibodies .

Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 (top panels) or PCNT (bottom panels). After 3-min MT regrowth, cells were fixed and stained with the indicated antibodies.


MT regrowth experiments in centrinone - treated akap KO cells transfected with either scramble or PCNT siRNAs . After a 3h NZ treatment , MTs were allowed to polymerize and , at the indicated time points after washout , cells were fixed and labelled for α - tubulin ( green ) , PCNT ( red ) and CAP350 ( blue ) .

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MT regrowth experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After a 3h NZ treatment, MTs were allowed to polymerize and, at the indicated time points after washout, cells were fixed and labelled for α-tubulin (green), PCNT (red) and CAP350 (blue).


MT repolymerization experiments in centrinone - treated akap KO cells transfected with either scramble or PCNT siRNAs . After NZ treatment and removal , cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin . Cells were finally fixed and triple stained with EB1 ( green ) , PCNT ( red ) and CAP350 ( blue ) antibodies . Enlarged views of the boxed areas are presented at right . Scale bars , 5 μm .

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MT repolymerization experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After NZ treatment and removal, cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin. Cells were finally fixed and triple stained with EB1 (green), PCNT (red) and CAP350 (blue) antibodies. Enlarged views of the boxed areas are presented at right. Scale bars, 5 μm.


Confocal images showing MT network in WT , akap KO , c5rap2 KO , pcnt KO and pc - ak - 2KO RPE - 1 cells fixed and stained with antibodies against α - tubulin ( left ) or EB1 ( right ) . The red and yellow lines indicate the contour of cells .

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Confocal images showing MT network in WT, akap KO, c5rap2 KO, pcnt KO and pc-ak-2KO RPE-1 cells fixed and stained with antibodies against α-tubulin (left) or EB1 (right). The red and yellow lines indicate the contour of cells.


Confocal images showing MT network in centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO backgrounds . Cells containing extra centrosomes induced by centrinone washout were also quantified ( bottom panels ) .

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Confocal images showing MT network in centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO backgrounds. Cells containing extra centrosomes induced by centrinone washout were also quantified (bottom panels).


Quantification of α - tubulin fluorescence intensity ( as a measure of MT mass polymer , C ) , in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean .

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Quantification of α-tubulin fluorescence intensity (as a measure of MT mass polymer, C), in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


Quantification of number of EB1 comets per cell ( D ) in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean .

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Quantification of number of EB1 comets per cell (D) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


Quantification of α , cell area ( E ) in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean .

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Quantification of α , cell area (E) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


Quantification of MT density ( MT mass polymer / cell area ) ( F ) in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean .

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Quantification of MT density (MT mass polymer/cell area) (F) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


A . Chemical structure of Nec - 1 . The schematic diagram was drawn by S . Lee using ChemDraw Professional 15 . 0 . software .

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A. Chemical structure of Nec-1. The schematic diagram was drawn by S. Lee using ChemDraw Professional 15.0. software.


B , C . MTT cytotoxicity assay . ( B ) HT22 cell line was treated with various concentrations of Nec - 1 ( 0 - 200 μM ) . Aggregated Aβ ( 1 - 42 ) was used as the positive control for the induction of neural cell death . All data represented the mean ± standard deviation of three independent experiments performed in triplicate .

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B, C. MTT cytotoxicity assay. (B) HT22 cell line was treated with various concentrations of Nec-1 (0 - 200 μM). Aggregated Aβ(1-42) was used as the positive control for the induction of neural cell death. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.


B , C . MTT cytotoxicity assay . ( C ) HT22 , BV2cell lines and primary astrocytes were pre - treated with Nec - 1 ( 50 μM ) 15 minutes prior to application of 10 μM of pre - aggregated Aβ ( 1 - 42 ) for indicated times . Cell proliferation was then measured by MTT assay . All data represented the mean ± standard deviation of three independent experiments performed in triplicate .

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B, C. MTT cytotoxicity assay. (C) HT22, BV2cell lines and primary astrocytes were pre-treated with Nec-1 (50 μM) 15 minutes prior to application of 10 μM of pre-aggregated Aβ(1-42) for indicated times. Cell proliferation was then measured by MTT assay. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.


D , E . Neural cell death levels measured by staining with the Live / Dead Viability / Cytotoxicity Assay Kit ( Molecular Probes ) . Pre - treatment of cells with 50 μM Nec - 1 preceded the application of 10 μM pre - aggregated Aβ ( 1 - 42 ) by 15 minutes . The assay was performed 24 hours after Aβ ( 1 - 42 ) application . ( D ) Representation of the stained cells . Scale bar = 500 μm ( E ) Quantification of dead cells stained by EthD - 1 .

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D, E. Neural cell death levels measured by staining with the Live/Dead Viability/Cytotoxicity Assay Kit (Molecular Probes). Pre-treatment of cells with 50 μM Nec-1 preceded the application of 10 μM pre-aggregated Aβ(1-42) by 15 minutes. The assay was performed 24 hours after Aβ(1-42) application. (D) Representation of the stained cells. Scale bar = 500 μm (E) Quantification of dead cells stained by EthD-1.


F . Internalization of Aβ ( 1 - 42 ) into HT22 and BV2 cell lines in the presence or the absence of Nec - 1 pre - treatment for indicated periods .

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F. Internalization of Aβ(1-42) into HT22 and BV2 cell lines in the presence or the absence of Nec-1 pre-treatment for indicated periods.


G . Cellular expression levels of indicated proteins in HT22 , BV2 cell lines , and primary astrocyte stimulated with Aβ ( 1 - 42 ) aggregates in the presence or the absence of Nec - 1 pre - treatment for 24 hours .

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G. Cellular expression levels of indicated proteins in HT22, BV2 cell lines, and primary astrocyte stimulated with Aβ(1-42) aggregates in the presence or the absence of Nec-1 pre-treatment for 24 hours.


A - E . Behavioural tests of APP / PS1mice . ( B - D ) Y - maze and passive avoidance tests on 7 - month - old APP / PS1mice after Nec - 1 administration for 12 weeks . Average alternation ( % ) for each test group ( B ) and total entry number into each arm ( C ) on Y - maze . ( D ) Average latency time in seconds for each test group on passive avoidance test .

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A-E. Behavioural tests of APP/PS1mice. (B-D) Y-maze and passive avoidance tests on 7-month-old APP/PS1mice after Nec-1 administration for 12 weeks. Average alternation (%) for each test group (B) and total entry number into each arm (C) on Y-maze. (D) Average latency time in seconds for each test group on passive avoidance test.


A - E . Behavioural tests of APP / PS1mice . ( E ) Survival of Wt and APP / PS1mice after injection of Nec - 1 ( 6 . 25 mg / kg ) .

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A-E. Behavioural tests of APP/PS1mice. (E) Survival of Wt and APP/PS1mice after injection of Nec-1 (6.25 mg/kg).


F - I . Behavrioural tests of Aβ ( 1 - 42 ) infusion mice . ( H , I ) Y - maze tests on Aβ ( 1 - 42 ) infusion mice model injected with Nec - 1 . Average alternation ( % ) for each test group ( H ) and total entry number into each arm ( I ) on Y - maze .

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F-I. Behavrioural tests of Aβ(1-42) infusion mice. (H, I) Y-maze tests on Aβ(1-42) infusion mice model injected with Nec-1. Average alternation (%) for each test group (H) and total entry number into each arm (I) on Y-maze.


A . Co - immunoprecipitation of RIPK3 with Aβ ( 1 - 42 ) in HT22 , BV2 cell lines , and primary astrocytes in response to Aβ ( 1 - 42 ) aggregates . IP , immunoprecipitation ; IB , immunoblot .

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A. Co-immunoprecipitation of RIPK3 with Aβ(1-42) in HT22, BV2 cell lines, and primary astrocytes in response to Aβ(1-42) aggregates. IP, immunoprecipitation; IB, immunoblot.


B . A close - up view of the interactions between Nec - 1 and surrounding residues in RIPK1 . Nec - 1 is shown in green . The hydrophobic residues around Nec - 1 in RIPK1 kinase domain are shown in sticks . Hydrogen bonds in this and all other figures are represented by yellow dashed lines . Docking score of Nec - 1 in RIPK1 was - 9 . 550 .

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B. A close-up view of the interactions between Nec-1 and surrounding residues in RIPK1. Nec-1 is shown in green. The hydrophobic residues around Nec-1 in RIPK1 kinase domain are shown in sticks. Hydrogen bonds in this and all other figures are represented by yellow dashed lines. Docking score of Nec-1 in RIPK1 was -9.550.


C . Surface treated view of 12 - mer Aβ fibrils ( 2LMO , VDW radius = 0 . 55 Å ) having hydrophobic pockets plausible for Nec - 1 binding .

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C. Surface treated view of 12-mer Aβ fibrils (2LMO, VDW radius = 0.55 Å) having hydrophobic pockets plausible for Nec-1 binding.


D . Docked structure of 12 - mer Aβ fibrils ( 2LMO ) with 4 molecules of Nec - 1 . Docking scores for four top - ranked Glide docking poses of Nec - 1 in Aβ plaques ; - 8 . 338 , - 4 . 755 , - 8 . 493 , and - 6 . 754 in clockwise order starting from the top - left corner .

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D. Docked structure of 12-mer Aβ fibrils (2LMO) with 4 molecules of Nec-1. Docking scores for four top-ranked Glide docking poses of Nec-1 in Aβ plaques; -8.338, -4.755, -8.493, and -6.754 in clockwise order starting from the top-left corner.


E . Surface plasmon resonance sensorgrams of Nec - 1 targeting Aβ ( 1 - 42 ) aggregates ( left ) , and the corresponding dissociation fitting curve from the saturated region ( right ) under various concentrations of Nec - 1 .

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E. Surface plasmon resonance sensorgrams of Nec-1 targeting Aβ(1-42) aggregates (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


4 - month - old APP / PS1 ( n = 13 , male ) and age - matched wild - type ( Wt , n = 14 , male ) mice were intravenously injected with Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for 12 weeks . After the completion of behavioural tests , the brains of each group of mice were subjected to analysis . A . ThS - stained insoluble Aβplaques in whole brain and the hippocampal region of each group . Scale bars = 1 mM ( upper ) , 200 μM ( lower ) . B - G . Statistics of ThS - positive Aβplaques in the brains of APP / PS1mice . Total numbers and areas of ThS - positive Aβplaques in the whole brains ( B and C ) , cortex ( D and E ) and hippocampus ( F and G ) .

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4-month-old APP/PS1 (n = 13, male) and age-matched wild-type (Wt, n = 14, male) mice were intravenously injected with Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for 12 weeks. After the completion of behavioural tests, the brains of each group of mice were subjected to analysis.A. ThS-stained insoluble Aβplaques in whole brain and the hippocampal region of each group. Scale bars = 1 mM (upper), 200 μM (lower).B-G. Statistics of ThS-positive Aβplaques in the brains of APP/PS1mice. Total numbers and areas of ThS-positive Aβplaques in the whole brains (B and C), cortex (D and E) and hippocampus (F and G).


A . Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild - type ( Wt ) and APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . Diffuse plaques in the brain sections were stained by anti - Aβ antibody ( 6E10 clone , green colour ) and anti - GFAP antibody ( red colour ) . Hoechst 33342 ( blue colour ) was applied for nuclear counterstaining . Scale bars = 200 μM .

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A. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). Diffuse plaques in the brain sections were stained by anti-Aβ antibody (6E10 clone, green colour) and anti-GFAP antibody (red colour). Hoechst 33342 (blue colour) was applied for nuclear counterstaining. Scale bars = 200 μM.


B - D . Dot blot analysis of total Aβ ( anti - Aβ : 6E10 clone , also recognizes APP ) and protein oligomer ( anti - amyloidogenic protein oligomer A11 ) in the cortical ( B ) and hippocampal ( C ) region of indicated groups . ( D ) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups . Relative intensity of each band was determined by densitometry of dot blots ( B and C ) using ImageJ software and normalized to vehicle - administered APP / PS1mouse group .

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B-D. Dot blot analysis of total Aβ (anti-Aβ: 6E10 clone, also recognizes APP) and protein oligomer (anti-amyloidogenic protein oligomer A11) in the cortical (B) and hippocampal (C) region of indicated groups. (D) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups. Relative intensity of each band was determined by densitometry of dot blots (B and C) using ImageJ software and normalized to vehicle-administered APP/PS1mouse group.


E . Sandwich ELISA of Aβ - soluble and - insoluble fractions from cortical and hippocampal lysates of APP / PS1mice .

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E. Sandwich ELISA of Aβ-soluble and -insoluble fractions from cortical and hippocampal lysates of APP/PS1mice.


F . Western blot analyses of cortical and hippocampal lysates from APP / PS1mice for APP and sAPP expressions .

F. Western blot analyses of cortical and hippocampal lysates from APP/PS1mice for APP and sAPP expressions.


G . ThT assays for the inhibition of Aβ aggregation . Fluorescence intensity was normalized to Aβaggregates ( 100 % , day 5 ) .

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G. ThT assays for the inhibition of Aβ aggregation. Fluorescence intensity was normalized to Aβaggregates (100%, day 5).


H . SDS - PAGE analysis of PICUP cross - linked Aβaggregates visualized by silver staining .

H. SDS-PAGE analysis of PICUP cross-linked Aβaggregates visualized by silver staining.


A . Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . The membranes analysed in ( A ) are identical to those in Fig 5F .

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A. Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). The membranes analysed in (A) are identical to those in Fig 5F.


B . Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild - type ( Wt ) and APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for phosphorylated tau expression levels . Anti - tau ( phospho Ser199 ) antibody ( red colour ) was applied for staining tau phosphorylation on Serine - 199 and Hoechst 33342 ( blue colour ) for nuclear counterstaining in the brain sections . Scale bars = 200 μM .

B. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for phosphorylated tau expression levels. Anti-tau (phospho Ser199) antibody (red colour) was applied for staining tau phosphorylation on Serine-199 and Hoechst 33342 (blue colour) for nuclear counterstaining in the brain sections. Scale bars = 200 μM.


C . Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec - 1 ( lower ) . The membranes analysed in ( C ) are identical to those in Fig 1G and 5F .

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C. Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec-1 (lower). The membranes analysed in (C) are identical to those in Fig 1G and 5F.


C . Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP / PS1mice injected with Nec - 1 ( upper ) . The membranes analysed in ( C ) are identical to those in Fig 1G and 5F .

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C. Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP/PS1mice injected with Nec-1 (upper). The membranes analysed in (C) are identical to those in Fig 1G and 5F.


D , E . ThT assays for the inhibition of tau aggregation ( D ) and for disaggregation of tau ( E ) . Fluorescence intensity was normalized to tauaggregates ( 100 % , day 5 ) .

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D, E. ThT assays for the inhibition of tau aggregation (D) and for disaggregation of tau (E). Fluorescence intensity was normalized to tauaggregates (100%, day 5).


F . Surface plasmon resonance sensorgrams of Nec - 1 targeting tau ( left ) , and the corresponding dissociation fitting curve from the saturated region ( right ) under various concentrations of Nec - 1 .

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F. Surface plasmon resonance sensorgrams of Nec-1 targeting tau (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and BMI Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) .

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and BMI Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression , plasma alanine aminotransferase ( ALAT ) ( B ) Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) .

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression , plasma alanine aminotransferase (ALAT) (B) Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and liver fat content ( C ) . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) .

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and liver fat content (C). Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).


ASK1 mRNA expression in patients with different NASH scores Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * *

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ASK1 mRNA expression in patients with different NASH scores Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG5 ( E ) mRNA expression . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * *

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG5 (E) mRNA expression. Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG7 Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) .

Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG7 Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).


Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG12 mRNA expression . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * *

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Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG12 mRNA expression. Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***


Hepatocytes were treated with BSA , Palm or Palm + Rap for 24 h . Cells were stained for lipid droplet accumulation ( Bodipy 493 / 503 , green ) and nuclei ( Hoechst , blue ) . Lipid accumulation was quantified using automated image - based analysis ( n = 4 biological replicates ) . Scale bar represents 100 µm .

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Hepatocytes were treated with BSA, Palm or Palm+Rap for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates). Scale bar represents 100 µm.


Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 ( siASK1 ) or non - targeting siRNA control ( siCtrl ) .

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Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 (siASK1) or non-targeting siRNA control (siCtrl).


Lipid accumulation was quantified using automated image - based analysis in ASK1 knockdown ( siASK1 ; grey bars ) or control ( siCtrl ; black bars ) cells treated with BSA , Palm , Palm + Rap and stained as mentioned above ( n = 4 biological replicates ) .

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Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm, Palm+Rap and stained as mentioned above (n=4 biological replicates).


Shown is one representative Western blot from two independent experiments and quantification of LC3 - II / I and p62 protein levels in siCtrl ( n = 4 biological replicates ) or siASK1 ( n = 4 biological replicates ) cells treated with Palm .

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Shown is one representative Western blot from two independent experiments and quantification of LC3-II/I and p62 protein levels in siCtrl (n=4 biological replicates) or siASK1 (n=4 biological replicates) cells treated with Palm.


Colocalization of LC3 - II punctate ( red ; arrow ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in siCtrl or siASK1 cells treated with BSA , Palm or Palm + Rap . Scale bar represents 100 µm .

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Colocalization of LC3-II punctate (red; arrow) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in siCtrl or siASK1 cells treated with BSA, Palm or Palm+Rap. Scale bar represents 100 µm.


Colocalization of LC3 - II punctate with lipid droplets ( BODIPY 493 / 503 , green ) was quantified in hepatocytes transfected with siRNA targeting ASK1 ( siASK1 ; grey bars ) or non - targeting siRNA control ( siCtrl ; black bars ) and treated with BSA or BSA + Baf for 24 hours ( n = 4 biological replicates ) .

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Colocalization of LC3-II punctate with lipid droplets (BODIPY 493/503, green) was quantified in hepatocytes transfected with siRNA targeting ASK1 (siASK1; grey bars) or non-targeting siRNA control (siCtrl; black bars) and treated with BSA or BSA+Baf for 24 hours (n=4 biological replicates).


Lipid accumulation was quantified using automated image - based analysis in ASK1 knockdown ( siASK1 ; grey bars ) or control ( siCtrl ; black bars ) cells treated with BSA , Palm or Palm + Baf and stained as mentioned above ( n = 4 biological replicates ) . Values are expressed as mean ± SEM . *

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Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm or Palm+Baf and stained as mentioned above (n=4 biological replicates). Values are expressed as mean ± SEM. *


Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm .

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Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.


Liver triglyceride ( TG ) content of mice fed a chow ( ASK1F / F n = 6 mice ; ASK1Δhep n = 6 mice ) or HFD ( ASK1F / F n = 14 mice ; ASK1Δhep n = 15 mice ) .

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Liver triglyceride (TG) content of mice fed a chow (ASK1F/F n=6 mice; ASK1Δhep n=6 mice) or HFD (ASK1F/F n=14 mice; ASK1Δhep n=15 mice).


Relative mRNA expression of perilipin ( Plin ) and very low - density lipoprotein receptor ( Vldlr ) in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( Vldlr ) or n = 8 mice ( Plin ) ) .

Relative mRNA expression of perilipin (Plin) and very low-density lipoprotein receptor (Vldlr) in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (Vldlr) or n=8 mice (Plin)).


Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( F4 / 80 , Mcp1 ) or ASK1Δhep n = 8 mice ( Il - 6 ) ) .

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Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (F4/80, Mcp1) or ASK1Δhep n=8 mice (Il-6)).


Relative mRNA expression of collagen , type I , alpha 1 ( Col1a1 ) ( ASK1F / F n = 11 mice ; ASK1Δhep n = 15 mice ) and transforming growth factor beta 1 ( Tgfβ1 ) ( ASK1F / F n = 6 mice ; ASK1Δhep n = 8 mice ) in liver of HFD - fed mice .

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Relative mRNA expression of collagen, type I, alpha 1 (Col1a1) (ASK1F/F n=11 mice; ASK1Δhep n=15 mice) and transforming growth factor beta 1 (Tgfβ1) (ASK1F/F n=6 mice; ASK1Δhep n=8 mice) in liver of HFD-fed mice.


Representative images of liver sections stained with Sirius Red ( scale bar represents 100 µm )

Representative images of liver sections stained with Sirius Red (scale bar represents 100 µm)


quantification of Sirius Red positive area in liver of mice fed a HFD ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) .

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quantification of Sirius Red positive area in liver of mice fed a HFD (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).


Representative images of liver sections stained with α - SMA from mice fed a HFD for 20 weeks . Scale bar represents 100 µm .

Representative images of liver sections stained with α-SMA from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.


Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) .

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Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).


Liver triglyceride ( TG ) content ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) and quantification of Sirius Red positive area in livers ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) of 15 months old chow - fed mice . Values are expressed as mean ± SEM . #

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Liver triglyceride (TG) content (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) and quantification of Sirius Red positive area in livers (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) of 15 months old chow-fed mice. Values are expressed as mean ± SEM. #


Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) .

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Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).


Representative immunofluorescence images of liver sections stained for LC3 - II punctate ( red ) and nuclei ( DAPI , blue ) in mice . Scale bar represents 100 µm .

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Representative immunofluorescence images of liver sections stained for LC3-II punctate (red) and nuclei (DAPI, blue) in mice. Scale bar represents 100 µm.


Primary hepatocytes isolated from chow - fed ASK1F / F ( ) and ASK1Δhep ( ) mice were treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf for 24 h . Cells were stained for lipid droplet accumulation ( Bodipy 493 / 503 , green ) and nuclei ( Hoechst , blue ) . Lipid accumulation was quantified using automated image - based analysis ( n = 4 biological replicates ) .

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Primary hepatocytes isolated from chow-fed ASK1F/F () and ASK1Δhep () mice were treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates).


Colocalization of LC3 - II punctate ( red ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in primary hepatocytes treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf . Scale bar represents 100 µm . Values are expressed as mean ± SEM .

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Colocalization of LC3-II punctate (red) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in primary hepatocytes treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf. Scale bar represents 100 µm. Values are expressed as mean ± SEM.


Western blots and quantification of respective protein levels in liver of HFD - fed mice treated with or without ASK1 inhibitor ( n = 4 mice per treatment ) .

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Western blots and quantification of respective protein levels in liver of HFD-fed mice treated with or without ASK1 inhibitor (n=4 mice per treatment).


Representative images of liver sections stained with H & E ( scale bar represents 100 µm )

Representative images of liver sections stained with H&E (scale bar represents 100 µm)


liver triglyceride ( TG ) content of HFD - fed mice treated with or without ASK1 inhibitor ( n = 8 mice per treatment ) .

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liver triglyceride (TG) content of HFD-fed mice treated with or without ASK1 inhibitor (n=8 mice per treatment).


Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm .

Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.


Liver triglyceride ( TG ) content ( ASK1F / F n = 11 mice ; ASK1 + hep n = 10 mice ) in livers of mice fed a HFD for 20 weeks .

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Liver triglyceride (TG) content (ASK1F/F n=11 mice; ASK1+hep n=10 mice) in livers of mice fed a HFD for 20 weeks.


Western blots and quantification of protein levels of respective targets in liver of HFD - fed mice ( n = 8 mice per genotype for pJNK and ATG12 , n = 4 mice per genotype for pBCN1 ) .

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Western blots and quantification of protein levels of respective targets in liver of HFD-fed mice (n=8 mice per genotype for pJNK and ATG12, n=4 mice per genotype for pBCN1).


Quantification of Sirius Red positive area ( ASK1F / F n = 11 mice ; ASK1 + hep n = 9 mice ) in livers of CCl4 - injected mice .

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Quantification of Sirius Red positive area (ASK1F/F n=11 mice; ASK1+hep n=9 mice) in livers of CCl4-injected mice.


Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM . #

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Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM. #


Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM .

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Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM.


Western blotting of endogenous Egg immunopurified from OSCs . Anti - Egg antibodies were used for immunoprecipitation ( IP ) and western blotting . Input : OSC total lysates used for IP . n . i . : nonimmune IgG used as an IP negative control . M . W . indicates protein molecular weight marker .

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Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP) and western blotting. Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control. M.W. indicates protein molecular weight marker.


Western blotting of Myc - Egg expressed in OSCs . Anti - Myc ( Myc ) and anti - β - Tubulin ( Tub ) antibodies were used . Tub was detected as a loading control . Control : empty vector was used as a negative control .

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Western blotting of Myc-Egg expressed in OSCs. Anti-Myc (Myc) and anti-β-Tubulin (Tub) antibodies were used. Tub was detected as a loading control. Control: empty vector was used as a negative control.


Western blotting of endogenous Egg immunopurified from OSCs . Anti - Egg antibodies were used for immunoprecipitation ( IP ) . Anti - Egg and anti - Ub antibodies were used for western blotting ( WB ) . Input : OSC total lysates used for IP . n . i . : nonimmune IgG used as an IP negative control .

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Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP). Anti-Egg and anti-Ub antibodies were used for western blotting (WB). Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control.


Western blotting of Egg immunopurified from total OSC lysates before ( - ) and after ( + ) phosphatase treatment . Anti - Egg antibodies were used for both immunoprecipitation ( IP ) and western blotting .

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Western blotting of Egg immunopurified from total OSC lysates before (-) and after (+) phosphatase treatment. Anti-Egg antibodies were used for both immunoprecipitation (IP) and western blotting.


Western blotting of Myc - Egg WT and its K1085R and S215 / T217 ( STAA ) mutants expressed in OSCs . Egg modifications identified in this study are summarized on the right . P : phosphorylation . Ub : monoubiquitination .

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Western blotting of Myc-Egg WT and its K1085R and S215/T217 (STAA) mutants expressed in OSCs. Egg modifications identified in this study are summarized on the right. P: phosphorylation. Ub: monoubiquitination.


In vitro histone methyltransferase assays . Top and second from the top : silver staining of purified Myc - Egg and Myc - EGFP . Second from the bottom : CBB staining of histone H3 . 1 . Bottom : 14C autoradiogram . EGFP : Myc - EGFP was used as a negative control . l . c . : light chain of antibody . Time : reaction time ( min ) of individual samples .

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In vitro histone methyltransferase assays. Top and second from the top: silver staining of purified Myc-Egg and Myc-EGFP. Second from the bottom: CBB staining of histone H3.1. Bottom: 14C autoradiogram. EGFP: Myc-EGFP was used as a negative control. l.c.: light chain of antibody. Time: reaction time (min) of individual samples.


Rescue assays . Myc - Egg WT and its K1085R and STAA mutants were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting ( WB ) was performed using anti - Egg and anti - Myc antibodies . RT - qPCR was used to quantify Stalker2 transposon expression level . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) . n . s . : not significant .

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Rescue assays. Myc-Egg WT and its K1085R and STAA mutants were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting (WB) was performed using anti-Egg and anti-Myc antibodies. RT-qPCR was used to quantify Stalker2 transposon expression level. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test). n.s.: not significant.


Immunofluorescence analysis of OSCs using anti - Egg antibodies . Endogenous Egg is shown in red . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

Immunofluorescence analysis of OSCs using anti-Egg antibodies. Endogenous Egg is shown in red. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Western blotting of fractionated OSC materials . Anti - Egg , anti - β - Tubulin ( Tub ) , and anti - histone H3 antibodies were used . H3 and Tub were detected as markers for nuclear ( Nuc ) and cytoplasmic ( Cyto ) fractions , respectively . Whole : the whole OSC lysates .

Western blotting of fractionated OSC materials. Anti-Egg, anti-β-Tubulin (Tub), and anti-histone H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.


Western blotting of Myc - Egg expressed in OSCs before ( siEGFP ) and after ( siUbc2 ) Ubc2 depletion . Anti - Myc - antibody was used for western blotting . The ratios of mUb - Egg over non - mUb - Egg band signals were 1 . 43 ( siEGFP ) and 0 . 78 ( siUbc2 ) .

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Western blotting of Myc-Egg expressed in OSCs before (siEGFP) and after (siUbc2) Ubc2 depletion. Anti-Myc-antibody was used for western blotting. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.43 (siEGFP) and 0.78 (siUbc2).


Immunofluorescence analysis using anti - Flag antibody to identify Flag - Ubc2 ( green ) expressed in OSCs . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

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Immunofluorescence analysis using anti-Flag antibody to identify Flag-Ubc2 (green) expressed in OSCs. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Immunofluorescence analysis of Myc - Egg WT , Myc - SV40 - NLS - Egg , and Myc - PKI - NES - Egg expressed in OSCs using anti - Myc antibody . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

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Immunofluorescence analysis of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs using anti-Myc antibody. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Western blotting of Myc - Egg WT , Myc - SV40 - NLS - Egg , and Myc - PKI - NES - Egg expressed in OSCs . Anti - Myc antibody was used . The ratios of mUb - Egg over non - mUb - Egg band signals were 1 . 50 ( WT ) , 2 . 29 ( SV40 - NLS ) , and 0 . 95 ( PKI - NES ) .

Western blotting of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs. Anti-Myc antibody was used. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.50 (WT), 2.29 (SV40-NLS), and 0.95 (PKI-NES).


Rescue assays . Myc - Egg WT , Myc - SV40 - NLS - Egg , and Myc - PKI - NES - Egg were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting was performed using anti - Egg ( left ) and anti - Myc ( right ) antibodies . Stalker2 transposon expression was quantified using RT - qPCR . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) .

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Rescue assays. Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. Stalker2 transposon expression was quantified using RT-qPCR. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).


In vitro HMT assays . Top : silver staining of purified Myc - Egg WT , Myc - Egg K1085R , Myc - SV40 - NLS - Egg WT , and Myc - SV40 - NLS - Egg K1085R . Middle : CBB staining of histone H3 . 1 . Bottom : 14C autoradiogram . Myc - EGFP was used as a negative control ( EGFP : not shown ) .

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In vitro HMT assays. Top: silver staining of purified Myc-Egg WT, Myc-Egg K1085R, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R. Middle: CBB staining of histone H3.1. Bottom: 14C autoradiogram. Myc-EGFP was used as a negative control (EGFP: not shown).


Rescue assays . Myc - Egg WT , Myc - Egg K1085R , and Myc - SV40 - NLS - K1085R were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting was performed using anti - Egg ( left ) and anti - Myc ( right ) antibodies . RT - qPCR quantified the expression level of the Stalker2 transposon . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) .

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Rescue assays. Myc-Egg WT, Myc-Egg K1085R, and Myc-SV40-NLS-K1085R were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of the Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).


Immunofluorescence analysis of endogenous Egg ( red ) in OSCs using anti - Egg antibody before ( siEGFP ) and after ( siWde ) Wde depletion . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

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Immunofluorescence analysis of endogenous Egg (red) in OSCs using anti-Egg antibody before (siEGFP) and after (siWde) Wde depletion. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Western blotting on fractionated materials of OSCs before ( siEGFP ) and after ( siWde ) Wde depletion . Anti - Egg , anti - Wde , anti - Tub , and anti - H3 antibodies were used . H3 and Tub were detected as markers for nuclear ( Nuc ) and cytoplasmic ( Cyto ) fractions , respectively . Whole : whole OSC lysates .

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Western blotting on fractionated materials of OSCs before (siEGFP) and after (siWde) Wde depletion. Anti-Egg, anti-Wde, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: whole OSC lysates.


Immunofluorescence analysis of endogenous Egg ( red ) in OSCs upon Flag - Wde ( green ) co - expression . Anti - Egg and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

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Immunofluorescence analysis of endogenous Egg (red) in OSCs upon Flag-Wde (green) co-expression. Anti-Egg and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Immunofluorescence analysis of Myc - Egg WT ( red ) in OSCs upon Wde co - expression ( green ) . Anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Wde co-expression (green). Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Western blotting on fractionated materials of Myc - Egg - expressed OSCs before ( - ) and after Wde WT and Wde ∆ CC co - expression . Anti - Myc , anti - Flag , anti - Tub , and anti - H3 antibodies were used . H3 and Tub were detected as markers for nuclear ( Nuc ) and cytoplasmic ( Cyto ) fractions , respectively . Whole : the whole OSC lysates .

Western blotting on fractionated materials of Myc-Egg-expressed OSCs before (-) and after Wde WT and Wde ∆CC co-expression. Anti-Myc, anti-Flag, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.


Protein - protein interaction assays . Myc - Egg and Flag - Wde WT and its ∆ CC mutant were expressed in OSCs . Immunoprecipitation was conducted using anti - Flag antibody while anti - Myc ( upper ) and anti - Flag ( lower ) antibodies were used for western blotting .

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Protein-protein interaction assays. Myc-Egg and Flag-Wde WT and its ∆CC mutant were expressed in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.


Immunofluorescence analysis of Myc - Egg WT ( red ) in OSCs upon Flag - Wde WT and ∆ CC mutant ( green ) co - expression . Anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Flag-Wde WT and ∆CC mutant (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Immunofluorescence analysis of Myc - Egg WT , Myc - Egg K987A , and Myc - Egg R988A mutants ( red ) in OSCs before and after Flag - Wde ( green ) co - expression . Anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

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Immunofluorescence analysis of Myc-Egg WT, Myc-Egg K987A, and Myc-Egg R988A mutants (red) in OSCs before and after Flag-Wde (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Western blotting of Myc - Egg WT and its K987A and R988A mutants . Flag - Wde : Flag - Wde was co - expressed . SV40 - NLS : SV40 - NLS was added to Myc - Egg . Anti - Myc and anti - Flag antibodies were used .

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Western blotting of Myc-Egg WT and its K987A and R988A mutants. Flag-Wde: Flag-Wde was co-expressed. SV40-NLS: SV40-NLS was added to Myc-Egg. Anti-Myc and anti-Flag antibodies were used.


Immunofluorescence analysis of Myc - Egg K987A and Myc - Egg R988A mutants ( red ) in OSCs . Both mutants were fused to SV40 - NLS . Anti - Myc antibody was used for detection . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

Immunofluorescence analysis of Myc-Egg K987A and Myc-Egg R988A mutants (red) in OSCs. Both mutants were fused to SV40-NLS. Anti-Myc antibody was used for detection. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Protein - protein interaction assays . Myc - Egg WT , Myc - SV40 - NLS - Egg WT , and Myc - SV40 - NLS - Egg K1085R mutant were expressed with Flag - Wde in OSCs . Immunoprecipitation was conducted using anti - Flag antibody while anti - Myc ( upper ) and anti - Flag ( lower ) antibodies were used for western blotting .

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Protein-protein interaction assays. Myc-Egg WT, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R mutant were expressed with Flag-Wde in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.


Rescue assays . Myc - Egg WT and Myc - SV40 - NLS - Egg WT were expressed in OSCs upon endogenous Egg and Wde depletion ( siEgg and siWde ) . Western blotting was performed using anti - Egg ( left ) and anti - Myc ( right ) antibodies . RT - qPCR quantified the expression level of Stalker2 transposon . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) .

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Rescue assays. Myc-Egg WT and Myc-SV40-NLS-Egg WT were expressed in OSCs upon endogenous Egg and Wde depletion (siEgg and siWde). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).


Western blotting of Myc - SV40 - NLS - Egg WT in whole ( Whole ) , cytoplasm / nucleoplasmic ( Cyto / Np ) and chromatin ( Chromatin ) fractions before ( - ) and after Flag - Wde co - expression . Anti - Myc and anti - Flag antibodies were used . Histone H3 was detected as a marker of chromatin fraction .

Western blotting of Myc-SV40-NLS-Egg WT in whole (Whole), cytoplasm/nucleoplasmic (Cyto/Np) and chromatin (Chromatin) fractions before (-) and after Flag-Wde co-expression. Anti-Myc and anti-Flag antibodies were used. Histone H3 was detected as a marker of chromatin fraction.


Immunofluorescence analysis of H3K9me3 ( green ) , Myc - Egg WT ( red ) , and Myc - SV40 - NLS - Egg WT ( red ) in OSCs before and after Flag - Wde ( yellow ) co - expression . Anti - H3K9me3 , anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

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Immunofluorescence analysis of H3K9me3 (green), Myc-Egg WT (red), and Myc-SV40-NLS-Egg WT (red) in OSCs before and after Flag-Wde (yellow) co-expression. Anti-H3K9me3, anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Box plots indicating the H3K9me3 signal intensities per cell related to Figure 5B ( p values based on unpaired Welch ' s t - test ) . Box plots show median ( line ) and 25th - 75th percentile ( box ) ± 1 . 5 interquartile range ( n > 25 cells for each box ) . Circles represent outliers .

Box plots indicating the H3K9me3 signal intensities per cell related to Figure 5B (p values based on unpaired Welch's t-test). Box plots show median (line) and 25th-75th percentile (box) ± 1.5 interquartile range (n > 25 cells for each box). Circles represent outliers.


Heat map of Egg ChIP - seq within a 10kb window centered on Egg peaks . Note that ChIP peaks was called using the ChIPed and Input samples Metaplots showing Egg ChIP - seq signals for genomic regions around Egg peaks . Displayed are fold - changes in Egg occupancy normalized to input .

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Heat map of Egg ChIP-seq within a 10kb window centered on Egg peaks. Note that ChIP peaks was called using the ChIPed and Input samples Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input.


Heatmaps of Egg ChIP - seq signal within 10 kb windows centered on Egg peaks in control ( siEGFP ) and Wde - depleted ( siWde ) OSCs .

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Heatmaps of Egg ChIP-seq signal within 10 kb windows centered on Egg peaks in control (siEGFP) and Wde-depleted (siWde) OSCs.


Metaplots showing Egg ChIP - seq signals for genomic regions around Egg peaks . Displayed are fold - changes in Egg occupancy normalized to input . Metapolts showing Egg ChIP - seq signals for genomic regions around transposon ( TE ) insertions .

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Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input. Metapolts showing Egg ChIP-seq signals for genomic regions around transposon (TE) insertions.


ChIP - qPCR was performed before ( siEGFP ) and after ( siWde ) Wde depletion . qPCR was performed for Piwi target transposons ( Stalker2 , mdg1 , and gypsy ) and normalized to % input of a PIwi non - target transposon ( roo ) . Error bars represent SD from three independent experiments .

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ChIP-qPCR was performed before (siEGFP) and after (siWde) Wde depletion. qPCR was performed for Piwi target transposons (Stalker2, mdg1, and gypsy) and normalized to %input of a PIwi non-target transposon (roo). Error bars represent SD from three independent experiments.


Density plots for Egg ChIP - seq signal on Stalker2 , mdg1 , and gypsy in control ( siEGFP ) and Wde - depleted ( siWde ) OSCs . Input : input DNA was used as a control . RPM : reads per million mapped reads .

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Density plots for Egg ChIP-seq signal on Stalker2, mdg1, and gypsy in control (siEGFP) and Wde-depleted (siWde) OSCs. Input: input DNA was used as a control. RPM: reads per million mapped reads.


G . Immunostain for Ki67 in wildtype and lymphoma - bearing spleen . Representative images are shown at left . The proportion of nuclei positive for Ki67 ( proliferation index ) is quantified at right for n = 3 spleens ( T - test p = 0 . 00002331 ) .

G. Immunostain for Ki67 in wildtype and lymphoma-bearing spleen. Representative images are shown at left. The proportion of nuclei positive for Ki67 (proliferation index) is quantified at right for n=3 spleens(T-test p=0.00002331).


F , G ( F ) PCR for Cγ1 - Cre and wildtype Cγ1 alleles and ( G ) , wildtype , floxed or excised Blimp1 alleles in DNA from wildtype ( wt ) control C57bl / 6J cells and purified T - cells or CD45 . 2 + cells from mouse with lymphoma ( T1 , T2 ) . PCR data were confirmed in at least 3 independent tests . Lad . = ladder .

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F, G (F) PCR for Cγ1-Cre and wildtype Cγ1 alleles and (G), wildtype, floxed or excised Blimp1 alleles in DNA from wildtype (wt) control C57bl/6J cells and purified T-cells or CD45.2+ cells from mouse with lymphoma (T1, T2). PCR data were confirmed in at least 3 independent tests. Lad. = ladder.


F . Western blot for c - MYC in whole spleen lysates from control C57bl / 6J and mPTCL mice . Quantification of c - MYC expression normalized to vinculin housekeeper gene levels ( n = 6 ) . T - test p = 0 . 0007 . Bars represent SEM .

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F. Western blot for c-MYC in whole spleen lysates from control C57bl/6J and mPTCL mice. Quantification of c-MYC expression normalized to vinculin housekeeper gene levels (n=6). T-test p=0.0007. Bars represent SEM.


B . Western blot for phosphorylated and total ZAP70 , AKT , S6 and GSK3β in lysates from three control ( C ) C57bl / 6J and three mPTCL ( T ) spleens . Murine DLBCL cell line A20 is a positive control for constitutively active AKT signaling .

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B. Western blot for phosphorylated and total ZAP70, AKT, S6 and GSK3β in lysates from three control (C) C57bl/6J and three mPTCL (T) spleens. Murine DLBCL cell line A20 is a positive control for constitutively active AKT signaling.


H . Western blot for γH2AX , total H2AX , CHK1 and RPA32 in control C57bl / 6J and mPTCL spleens . GAPDH is a loading control . Three representative samples per group are shown in the blot ( left ) and quantification was performed on n = 6 at right . T - test p = 0 . 0798 ( CHK1 ; not statistically significant , ns ) , p = 8 . 69E - 05 ( RPA32 ) , p = 2 . 11E - 03 ( γH2AX ) . * * * p < 0 . 001 .

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H. Western blot for γH2AX, total H2AX, CHK1 and RPA32 in control C57bl/6J and mPTCL spleens. GAPDH is a loading control. Three representative samples per group are shown in the blot (left) and quantification was performed on n=6 at right. T-test p=0.0798 (CHK1; not statistically significant, ns), p=8.69E-05 (RPA32), p=2.11E-03 (γH2AX). ***p<0.001.


A . Sanger screen IC50 drug sensitivity data ( IC50 values ) for DDR inhibitors targeting ATR , CHK1 / 2 and Wee1 in T - ALL , PTCL ( DEL , KARPAS - 299 , SUP - M2 ) and pan - cancer cell lines . ANOVA p = 0 . 413 ( AZD6738 , ATR inhibitor ) , p = 0 . 750 ( AZD772 , CHK1 / 2 inhibitor ) , p = 0 . 119 ( MK - 8776 , ATR inhibitor ) and 0 . 282 ( AZD1775 , Wee1 inhibitor ) . Bars represent SEM of biological replicates .

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A. Sanger screen IC50 drug sensitivity data (IC50 values) for DDR inhibitors targeting ATR, CHK1/2 and Wee1 in T-ALL, PTCL (DEL, KARPAS-299, SUP-M2) and pan-cancer cell lines. ANOVA p=0.413 (AZD6738, ATR inhibitor), p=0.750 (AZD772, CHK1/2 inhibitor), p=0.119 (MK-8776, ATR inhibitor) and 0.282 (AZD1775, Wee1 inhibitor). Bars represent SEM of biological replicates.


B . PTCL ( red ) and T - ALL ( yellow ) human cell lines were dosed in vitro with AZD6738 for 72h and viable cell numbers assayed by luminescence readout using Cell TiterGlo . Average IC50 values from three experiments are plotted . Bars represent SEM .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O 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. PTCL (red) and T-ALL (yellow) human cell lines were dosed in vitro with AZD6738 for 72h and viable cell numbers assayed by luminescence readout using Cell TiterGlo. Average IC50 values from three experiments are plotted. Bars represent SEM.


F . Western blot for γH2AX relative to total H2AX levels of spleen lysates from mice dosed for 71 days with AZD6738 ( ATR ; n = 4 ) relative to untreated mPTCL spleens taken at endpoint ( n = 6 ) . T - test p = 2 . 70E - 06 . PC9 cells dosed in vitro 24h with 1 µM aphidicolin ( Aph ; an inhibitor of DNA replication ) or with AZD6738 ( ATR ) are positive controls for DNA damage . Bars represent SEM . * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

F. Western blot for γH2AX relative to total H2AX levels of spleen lysates from mice dosed for 71 days with AZD6738 (ATR; n=4) relative to untreated mPTCL spleens taken at endpoint (n=6). T-test p=2.70E-06. PC9 cells dosed in vitro 24h with 1 µM aphidicolin (Aph; an inhibitor of DNA replication) or with AZD6738 (ATR) are positive controls for DNA damage. Bars represent SEM. ***p<0.001


( C ) Figures show the presence of differentially expressed genes within the analyzed single cells . Black color indicates cells with RPKM values above 40 for Pax3 , 300 for Stmn2 , 200 for Slc1a3 and 100 for

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

(C) Figures show the presence of differentially expressed genes within the analyzed single cells. Black color indicates cells with RPKM values above 40 for Pax3, 300 for Stmn2, 200 for Slc1a3 and 100 for Mbp


( B ) Expression ( RPKM ) of Sox3 and Sox9 in NPC and GPC cultures from RNA - seq experiments . Results are represented as mean ± SEM from 3 - 4 experiments ( n = 3 for NPC and n = 4 for GPC , * * * equals p = 4x10 - 5 , t . test )

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(B) Expression (RPKM) of Sox3 and Sox9 in NPC and GPC cultures from RNA-seq experiments. Results are represented as mean ± SEM from 3-4 experiments (n=3 for NPC and n=4 for GPC, *** equals p=4x10-5, t.test)


( C ) Immunohistochemical analysis of SOX3 and SOX9 expression in ESC derived NPC and GPC cultures . Analysis of TUJ1 , GFAP and SOX10 protein expression in differentiated cultures revealed that NPC give rise to neurons and GPCs to astrocytes and oligodendrocytes . Scale bar represents

(C) Immunohistochemical analysis of SOX3 and SOX9 expression in ESC derived NPC and GPC cultures. Analysis of TUJ1, GFAP and SOX10 protein expression in differentiated cultures revealed that NPC give rise to neurons and GPCs to astrocytes and oligodendrocytes. Scale bar represents 40µm


1 ] . ( F ) H3K27Ac read density in NPCs or GPCs around DNA - regions bound by SOX3 in NPCs . SOX3 DNA - regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci . Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II

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1]. (F) H3K27Ac read density in NPCs or GPCs around DNA-regions bound by SOX3 in NPCs. SOX3 DNA-regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci. Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II loci


( G ) Expression pattern of genes associated with group I and II loci ( from Fig . 2E ) within differentially expressed gene sets . Significance calculated by prop . test R , ( * * * ) P < 0 . 001 . ( H ) Venn diagram shows overlap between SOX3 binding in NPCs and GPCs . Bar graph shows expression pattern of genes continuously bound by SOX3 NPCs and GPCs . ( I ) Venn diagram shows overlap between SOX3 and SOX9 binding in GPCs . Bar graph shows expression pattern of genes co - bound by SOX3 and SOX9 in GPCs . ( J ) ChIP - seq peak graphics around the astrocyte gene Fgfbp3 . ChIP - seq peaks are derived from three different experiments ; SOX3 ChIPs in NPCs , SOX3 ChIPs in GPCs , SOX9 ChIPs in GPCs . Both ChIP - seq reads and called peak regions ( underlying black lines ) are shown for all data sets . Bar graphs shows the distribution of differentially expressed genes that are bound by all three factors . P - values ( phyper , R ) were calculated from the total number of protein coding genes in mm10 assembly ( 23 ´ 389 )

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(G) Expression pattern of genes associated with group I and II loci (from Fig. 2E) within differentially expressed gene sets. Significance calculated by prop.test R, (***) P<0.001. (H) Venn diagram shows overlap between SOX3 binding in NPCs and GPCs. Bar graph shows expression pattern of genes continuously bound by SOX3 NPCs and GPCs. (I) Venn diagram shows overlap between SOX3 and SOX9 binding in GPCs. Bar graph shows expression pattern of genes co-bound by SOX3 and SOX9 in GPCs. (J) ChIP-seq peak graphics around the astrocyte gene Fgfbp3. ChIP-seq peaks are derived from three different experiments; SOX3 ChIPs in NPCs, SOX3 ChIPs in GPCs, SOX9 ChIPs in GPCs. Both ChIP-seq reads and called peak regions (underlying black lines) are shown for all data sets. Bar graphs shows the distribution of differentially expressed genes that are bound by all three factors. P-values (phyper, R) were calculated from the total number of protein coding genes in mm10 assembly (23´389)


( A ) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA -

(A) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA-seq


( B ) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets . Yellow bar represents enrichment within NPC / GPC genes , green bar within neuronal genes , blue bar within astrocytic genes and red bar within oligodendrocytic genes . SOX10 ChIP - seq data from [

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(B) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets. Yellow bar represents enrichment within NPC/GPC genes, green bar within neuronal genes, blue bar within astrocytic genes and red bar within oligodendrocytic genes. SOX10 ChIP-seq data from [37


( F ) Expression of Nfia , Ehf and Zbtb3 among spinal cord cells analyzed with scRNA - seq . Red color indicates cells with RPKM values above 55 for Nfia , 5 for Zbtb3 and 5 for Ehf . n = 3 for SOX9 and 2 for SOX3 . P - values ( phyper , R ) were calculated from the total number of genes for mm10 ( 23 ´ 389 )

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

(F) Expression of Nfia, Ehf and Zbtb3 among spinal cord cells analyzed with scRNA-seq. Red color indicates cells with RPKM values above 55 for Nfia, 5 for Zbtb3 and 5 for Ehf. n=3 for SOX9 and 2 for SOX3. P-values (phyper, R) were calculated from the total number of genes for mm10 (23´389)


( A ) Luc - reporter assays in P19 cells . Luc - reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA ; an activation that could be efficiently blocked by SOX3 . No activation of these reporters could be detected with SOX10 or SOX11 . ( B ) Luc - reporters containing SOX9 and SOX10 bound DNA - regions around the astrocyte genes Aldh1l1 and Atp1a2 , which are not expressed in SOX10 + oligodendrocytes , could not be activated by SOX9 or SOX10

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(A) Luc-reporter assays in P19 cells. Luc-reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA; an activation that could be efficiently blocked by SOX3. No activation of these reporters could be detected with SOX10 or SOX11. (B) Luc-reporters containing SOX9 and SOX10 bound DNA-regions around the astrocyte genes Aldh1l1 and Atp1a2, which are not expressed in SOX10+ oligodendrocytes, could not be activated by SOX9 or SOX10


( C ) Luc - reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10 . No activation could be detected with SOX11 . ( D ) Luc - reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2 , which are sequentially bound by SOX3 and SOX11 , could be activated by SOX11 and efficiently repressed by SOX3 . No activation could be detected with

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(C) Luc-reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10. No activation could be detected with SOX11. (D) Luc-reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2, which are sequentially bound by SOX3 and SOX11, could be activated by SOX11 and efficiently repressed by SOX3. No activation could be detected with SOX10


( E ) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes . Scale bar , 25µm . ( F ) Statistical view of Fig . 5E . Bars represents mean values ( of 3 experiments ) of cells expressing GFAP or SOX10 in GFP or Sox3 - transduced cells . Error bars in figure 5 indicate standard deviation of 3 - 7 individual samples and three - star significance > 0 . 001 ( t - test ) . ( * ) 0 , 05 < P < 0 , 01 ; ( * * ) 0 , 01 < P < 0 , 001 ; ( * * * ) P < 0 ,

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(E) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes. Scale bar, 25µm. (F) Statistical view of Fig. 5E. Bars represents mean values (of 3 experiments) of cells expressing GFAP or SOX10 in GFP or Sox3-transduced cells. Error bars in figure 5 indicate standard deviation of 3-7 individual samples and three-star significance > 0.001 (t-test). (*) 0,05<P<0,01; (**) 0,01<P<0,001; (***) P<0,001


( A ) SOX3 over - expression experiments in chicken spinal cord at E4 . 48 hrs post electroporation , transfected side showed decreased expression of FGFR2 , NFIA and Fgfr3 . Bar : 50µm . ( B ) 24 hrs after miss - expression of a dominant negative version of SOXB1 ( dnSOXB1 ) , premature expression of FGFR2 , NFIA and Fgfr3 were found in the transfected side . Bar : 40µm . ( C ) 24 hrs after SOX9 over - expression in chicken spinal cord ( at E4 ) , increased expression of FGFR2 , NFIA and Fgfr3 was shown compared to the un - transfected control side . Bar : 40µm . ( D ) Sox3 and Sox9 co - electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2 , NFIA and Fgfr3 levels were comparable to the un - transfected control side . Embryos were analyzed 24 hrs post electroporation . Bar : 40µm . ( E

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

(A) SOX3 over-expression experiments in chicken spinal cord at E4. 48 hrs post electroporation, transfected side showed decreased expression of FGFR2, NFIA and Fgfr3. Bar: 50µm. (B) 24 hrs after miss-expression of a dominant negative version of SOXB1 (dnSOXB1), premature expression of FGFR2, NFIA and Fgfr3 were found in the transfected side. Bar: 40µm. (C) 24 hrs after SOX9 over-expression in chicken spinal cord (at E4), increased expression of FGFR2, NFIA and Fgfr3 was shown compared to the un-transfected control side. Bar: 40µm. (D) Sox3 and Sox9 co-electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2, NFIA and Fgfr3 levels were comparable to the un-transfected control side. Embryos were analyzed 24 hrs post electroporation. Bar: 40µm. (E


( B ) Immunoblotting of whole cell extracts obtained from parental and RAP80 - / - clones in the RPE - 1 hTERT p53 - / - Cas9 , RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 , U2OS , U2OS - 2 - 6 - 3 and U2OS Flp - In / TREx cell lines with RAP80 antibodies . Tubulin is used as loading control . Representative of at least two independent immunoblots . See Figure EV1 for further details on the gene editing strategy used to knockout RAP80 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblotting of whole cell extracts obtained from parental and RAP80-/- clones in the RPE-1 hTERT p53-/- Cas9, RPE-1 hTERT p53-/- BRCA1-/- Cas9, U2OS, U2OS-2-6-3 and U2OS Flp-In/TREx cell lines with RAP80 antibodies. Tubulin is used as loading control. Representative of at least two independent immunoblots. See Figure EV1 for further details on the gene editing strategy used to knockout RAP80.


( C , D ) The indicated parental and RAP80 - / - cell lines were processed for immunofluorescence 1 h post - irradiation ( 10 Gy ) and stained with antibodies against ABRAXAS1 and γH2AX . Shown in ( C ) is the percentage of cells with > 5 ABRAXAS1 foci that colocalize with γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates ) . Representative micrographs of RPE1 - hTERT p53 - / - Cas9 wild type ( WT ) and RAP90 - / - cells are shown in ( D ) .

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(C, D) The indicated parental and RAP80-/- cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against ABRAXAS1 and γH2AX. Shown in (C) is the percentage of cells with > 5 ABRAXAS1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs of RPE1-hTERT p53-/- Cas9 wild type (WT) and RAP90-/- cells are shown in (D).


( E , The indicated cell lines were processed for immunofluorescence 1 h post - irradiation ( 10 Gy ) and stained with antibodies against BRCA1 and γH2AX . Shown in ( E ) is the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates for all conditions except n = 5 for U2OS WT , n = 2 for U2OS 2 - 6 - 3 cells , n = 7 , 5 , 4 for U2OS Flp - In / T - REx cells ) .

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(E, The indicated cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against BRCA1 and γH2AX. Shown in (E) is the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates for all conditions except n=5 for U2OS WT, n=2 for U2OS 2-6-3 cells, n=7, 5, 4 for U2OS Flp-In/T-REx cells).


( G ) RPE - 1 hTERT p53 - / - Cas9 parental ( WT ) and RAP80 - / - cells were untreated ( t = 0 h ) or irradiated with a 10 Gy dose . Samples were collected at the indicated time points , processed for immunofluorescence and stained with antibodies against BRCA1 and γH2AX . Shown is the percentage of cells with > 5 BRCA1 foci . A minimum of 100 cells per replicate were analyzed and the datapoints represent the mean ± S . D . ( n = 3 biological replicates ) . All scale bars are 5 μm .

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(G) RPE-1 hTERT p53-/- Cas9 parental (WT) and RAP80-/- cells were untreated (t=0 h) or irradiated with a 10 Gy dose. Samples were collected at the indicated time points, processed for immunofluorescence and stained with antibodies against BRCA1 and γH2AX. Shown is the percentage of cells with > 5 BRCA1 foci. A minimum of 100 cells per replicate were analyzed and the datapoints represent the mean ± S.D. (n=3 biological replicates). All scale bars are 5 μm.


( A , B ) U2OS Flp - In / T - Rex parental ( WT ) and RAP80 - / - cells were transfected with siRNA pools targeting BRCA1 , RNF8 , RNF168 or BARD1 or with a non - targeting control siRNA ( CTRL ) . 48 h post - transfection cells were irradiated ( 2 Gy ) and processed for immunofluorescence 1 h post - IR treatment using antibodies against BRCA1 and γH2AX . Quantitation of the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX is shown in ( A ) . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates ) . Representative micrographs are shown in ( B ) .

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

(A, B) U2OS Flp-In/T-Rex parental (WT) and RAP80-/- cells were transfected with siRNA pools targeting BRCA1, RNF8, RNF168 or BARD1 or with a non-targeting control siRNA (CTRL). 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX is shown in (A). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (B).