A . thaliana seedling ( J ) target gene mRNA induction ( n = 3 I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD ) Student ' s t -

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A. thaliana seedling (J) target gene mRNA induction (n=3 I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD) Student's t-tes


A . thalian leaf piece ( K ) ROS production ( 0 - 45 min , n = 6 ) Data information represent data ( mean + SEM ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate plant

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A. thalian leaf piece (K) ROS production (0-45 min, n=6) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate plant leave


TNF released from WT or Myd88 ( A KO BMDM ( total n = 6 - 9 / group combined from 3 experiments ) Data information : I represent data ( mean + SEM ) combined from ' n ' technical or biological replicate mic Student ' t -

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TNF released from WT or Myd88 (A KO BMDM (total n=6-9/group combined from 3 experiments) Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate mic Student' t-test


TNF released from W or Tlr KO BMDM ( total n = 6 - 9 / group combined from 3 experiments ) Student ' s t -

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TNF released from W or Tlr KO BMDM (total n=6-9/group combined from 3 experiments) Student's t-tes


( C ) IL - 6 released from primary MoMacs ( n = 5 ) treated with non - targeting ( NT ) , TLR2 - or MyD88 - specific siRNA relative to NT condition one - sample t -

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(C) IL-6 released from primary MoMacs (n=5) treated with non-targeting (NT), TLR2- or MyD88-specific siRNA relative to NT condition one-sample t-tes


( D ) CD62L shedding from human PMNs ( n = 4 - 8 ) with or without isotype control or anti - TLR2 blocking antibody Data information : I represent data ( mean + SEM ) combined from ' n ' technical or biological replicate ( human donor * p < 0 . 05 according to Mann -

O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

(D) CD62L shedding from human PMNs (n=4-8) with or without isotype control or anti-TLR2 blocking antibody Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate (human donor * p<0.05 according to Mann-Whitney


( E ) IL - 6 protein released from mock or TLR2 Cas9 - CRISPR - edited THP - 1 cells ( n = 3 ) I one representative of ' n ' independent experiments is shown ( mean + SD ) ) , Student ' s t -

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(E) IL-6 protein released from mock or TLR2 Cas9-CRISPR-edited THP-1 cells (n=3) I one representative of 'n' independent experiments is shown (mean+SD) ), Student's t-tes


( F ) NF - κB activity in empty vector ( EV ) or TLR2 - transfected HEK293T cells ( n = 3 ) I one representative of ' n ' independent experiments is shown ( mean + SD )

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(F) NF-κB activity in empty vector (EV) or TLR2-transfected HEK293T cells (n=3) I one representative of 'n' independent experiments is shown (mean+SD)


( G ) MPO release from intradermally injected WT or Tlr2 KO mice ( n = 5 - 15 biopsies each ) Data information I represent data ( mean + SEM ) combined from ' n ' technical or biological replicate mic ) , Student ' s t -

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(G) MPO release from intradermally injected WT or Tlr2 KO mice (n=5-15 biopsies each) Data information I represent data (mean+SEM) combined from 'n' technical or biological replicate mic ), Student's t-tes


( H ) BALF IL - 6 and TNF from intratracheally treated WT or Tlr2 KO mice ( n = 4 - 5 mice each ) one - way ANOVA with Dunnett ' s multiple compariso

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(H) BALF IL-6 and TNF from intratracheally treated WT or Tlr2 KO mice (n=4-5 mice each) one-way ANOVA with Dunnett's multiple compariso


( I ) Relative IL10 mRNA in whole blood of WT or heterozygous TLR2 R753Q carriers ( n = 5 each ) Data information : I represent data ( mean + SEM ) combined from ' n ' technical or biological replicate ( human donor * p < 0 . 05 according to Mann -

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(I) Relative IL10 mRNA in whole blood of WT or heterozygous TLR2 R753Q carriers (n=5 each) Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate (human donor * p<0.05 according to Mann-Whitney


( A ) NF - κB activity in TLR2 - transfected HEK293T cells ( n = 3 ) Data information I one representative of ' n ' independent experiments is shown ( mean + SD ) Student ' s t -

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(A) NF-κB activity in TLR2-transfected HEK293T cells (n=3) Data information I one representative of 'n' independent experiments is shown (mean+SD) Student's t-tes


( B ) IL - 8 release and ROS production from primary PMNs ( n = 7 ) Data information represent data ( mean + SEM ) from ' n ' technical or biological replicates ( donors ) * p < 0 . 05 according to Wilcoxon signed rank

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

(B) IL-8 release and ROS production from primary PMNs (n=7) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) * p<0.05 according to Wilcoxon signed rank su


( D ) IRAK4 S186 and T345 phospho - site containing IRAK4 peptides quantified in proteome - wide phospho - screen in THP - 1 cells ( n = 1 ) Data informatio I one representative of ' n ' independent experiments is shown ( mean + SD ) for details on data acquisition and analysis for panel see Expanded View

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

(D) IRAK4 S186 and T345 phospho-site containing IRAK4 peptides quantified in proteome-wide phospho-screen in THP-1 cells (n=1) Data informatio I one representative of 'n' independent experiments is shown (mean+SD) for details on data acquisition and analysis for panel see Expanded View Information


( F ) Microarray intensities ( excerpt ) of chitin - specific signature genes ( n = 5 ) Data information represent data ( mean + SEM ) from ' n ' technical or biological replicates ( donors ) for details on data acquisition and analysis for panels ( D - F ) see Expanded View

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Microarray intensities (excerpt) of chitin-specific signature genes (n=5) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) for details on data acquisition and analysis for panels (D-F) see Expanded View Information


( G ) Relative cytokine mRNAs in whole blood stimulation ( n = 5 - 13 ) Data information represent data ( mean + SEM ) from ' n ' technical or biological replicates ( donors ) * p < 0 . 05 according to Wilcoxon signed rank

O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Relative cytokine mRNAs in whole blood stimulation (n=5-13) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) * p<0.05 according to Wilcoxon signed rank su


Flow cytometric quantificatio of Alexa647 - labeled C10 - 15 interaction with mTLR2 - Fc Protein ( n = 4 each ) Data information : Quantification sub - panel represent data ( mean + SE combined from ' n ' biological replicate mic I one representative of ' n ' independent experiments is show p < 0 . 05 according to Mann - Whitney U (

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

Flow cytometric quantificatio of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show p<0.05 according to Mann-Whitney U (A


microscale thermophoresis ( MST ) analysis ( B ) of Alexa647 - labeled C10 - 15 interaction with mTLR2 - Fc Protein ( n = 4 each ) I one representative of ' n ' independent experiments is

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

microscale thermophoresis (MST) analysis (B) of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) I one representative of 'n' independent experiments is show


( C ) Flow cytometr of Candida albicans stained with control IgG or mTLR2 - Fc anti - Fc - Alexa594 , together with ConA - Alexa488 and CFW ( n = 4 each ) . Scale bar = 5 μm Data information : Quantification sub - panel represent data ( mean + SE combined from ' n ' biological replicate mic I one representative of ' n ' independent experiments is show one - sample t - test (

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

(C) Flow cytometr of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show one-sample t-test (C


( D ) fluorescence microscopy of Candida albicans stained with control IgG or mTLR2 - Fc anti - Fc - Alexa594 , together with ConA - Alexa488 and CFW ( n = 4 each ) . Scale bar = 5 μm I one representative of ' n ' independent experiments is

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

(D) fluorescence microscopy of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm I one representative of 'n' independent experiments is show


( g ) MST analysis of Alexa647 - labeled C10 - 15 and mTLR2 - Fc protein with SSL3 titration ( n = 2 ) Data information I one representative of ' n ' independent experiments is

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

(g) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc protein with SSL3 titration (n=2) Data information I one representative of 'n' independent experiments is show


( H ) NF - κB activation in TLR2 - transfected HEK293T cells ( n = 3 Data information I one representative of ' n ' independent experiments is shown ( for asmean + SD ) ) , Student ' s t -

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) NF-κB activation in TLR2-transfected HEK293T cells (n=3 Data information I one representative of 'n' independent experiments is shown (for asmean+SD) ), Student's t-tes


IL - 8 release from primary PMNs ( n = 2 ) without or with SSL3 Data information I one representative of ' n ' independent experiments is show asmean + SD ) Student ' s t -

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IL-8 release from primary PMNs (n=2) without or with SSL3 Data information I one representative of 'n' independent experiments is show asmean+SD) Student's t-tes


( J ) BALF TNF in C57BL / 6 mice ( n = 5 / group ) upon C10 - 15 administration without or with SSL3 Data information : Quantificatio - panel represent data ( mean + SE combined from ' n ' biological replicate mic one - way ANOVA with Dunnett ' s multiple comparison ( J )

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

(J) BALF TNF in C57BL/6 mice (n=5/group) upon C10-15 administration without or with SSL3 Data information: Quantificatio -panel represent data (mean+SE combined from 'n' biological replicate mic one-way ANOVA with Dunnett's multiple comparison (J)


( K ) ROS production between 0 and 45 min post chitin application in A . thaliana leaf pieces ( n = 6 ) Data information : Quantificatio panels ( represent dat mean ± SD in K combined from ' n ' biological replicate plant

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

(K) ROS production between 0 and 45 min post chitin application in A. thaliana leaf pieces (n=6) Data information: Quantificatio panels ( represent dat mean±SD in K combined from 'n' biological replicate plant leave


( L ) MST analysis of Alexa647 - labeled C10 - 15 and mTLR2 - Fc Protein with C5 titration ( n = 2 ) Data informatio I one representative of ' n ' independent experiments is

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

(L) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc Protein with C5 titration (n=2) Data informatio I one representative of 'n' independent experiments is show


B . Screening results of compounds competing Dvl - CXXC5 interaction . Each 2 μM of the compounds was used for competing as described in Fig 1A . [ n = 3 ]

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

B . Screening results of compounds competing Dvl-CXXC5 interaction. Each 2 μM of the compounds was used for competing as described in Fig 1A. [n=3]


C , D . Primary calvaria cells were treated with 2 μM of each compound for 2 days . The cells were subjected to immunofluorescence analyses to visualize β - catenin ( C ; green ) . The relative numbers of β - catenin - positive nuclei were counted ( D ) . [ n = 3 ]

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C, D. Primary calvaria cells were treated with 2 μM of each compound for 2 days. The cells were subjected to immunofluorescence analyses to visualize β-catenin (C; green). The relative numbers of β-catenin-positive nuclei were counted (D). [n=3]


E , F . The calvariae from 4 - day - old mice were cultured ex vivo for 7 days with 2 µM of each compound . Representative calvaria sections were visualized by H & amp ; amp ; E staining ( E ) . The calvaria thicknesses were measured from the images using Image Pro software ( F ) . [ n = 3 ]

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E, F . The calvariae from 4-day-old mice were cultured ex vivo for 7 days with 2 µM of each compound. Representative calvaria sections were visualized by H&amp;E staining (E). The calvaria thicknesses were measured from the images using Image Pro software (F). [n=3]


B . A competition curve for the Dvl - CXXC5 interaction by KY - 02061 .

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B . A competition curve for the Dvl-CXXC5 interaction by KY-02061.


C - E . NMR titration analyses for Dvl PZD domain with KY - 02061 . 1H - 15N - HSQC analyses were performed to analyze the interaction of 15N - labelled Dvl - PDZ domain with KY - 02061 . The 1H - 15N - HSQC spectrum of different molar ratios ( Dvl PDZ domain : KY - 02061 ) are displayed as red ( 1 : 0 ) , orange ( 1 : 10 ) , purple ( 1 : 20 ) , cyan ( 1 : 40 ) , green ( 1 : 60 ) and blue ( 1 : 80 ) ( C , residues with meaningful chemical shift change are indicated by arrows ) . Plot of chemical shift changes ( Δδ ) as a function of residue number in molecular ratio 1 : 80 ( D , a red - colored line indicates the line for Δδ = 0 . 05 ) . The residues with Δδ greater than 0 . 05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure ( E ) .

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

C-E. NMR titration analyses for Dvl PZD domain with KY-02061. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02061. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02061) are displayed as red (1:0), orange (1:10), purple (1:20), cyan (1:40), green (1:60) and blue (1:80) (C, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:80 (D, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (E).


F . Molecular docking of Dvl binding motif ( DBM ) or KY - 02061 to Dvl PDZ domain were analyzed by in silico experiments . The superimposed structure of DBM ( green ) and KY - 02061 ( yellow ) on the surface of Dvl PDZ domain ( gray ) was visualized .

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

F. Molecular docking of Dvl binding motif (DBM) or KY-02061 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of DBM (green) and KY-02061 (yellow) on the surface of Dvl PDZ domain (gray) was visualized.


A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV - β - gal . After 24 h , the cells were treated with DMSO , KY - 02061 in DMSO , or 5 µM PolyR - DBM for 2 days . The luciferase activities of whole cell lysates were measured and normalized with β - galactosidase activities . [ n = 3 ]

O O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with DMSO, KY-02061 in DMSO, or 5 µM PolyR-DBM for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]


B . Primary calvaria cells were isolated from the calvariae of 4 - day - old mice ( Manton et al , 2007 ) , and treated with DMSO or KY - 02061 in DMSO for 4 days . ALP activity levels were visualized by ALP staining .

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B . Primary calvaria cells were isolated from the calvariae of 4-day-old mice (Manton et al, 2007), and treated with DMSO or KY-02061 in DMSO for 4 days. ALP activity levels were visualized by ALP staining.


C . Primary calvaria cells were treated with DMSO or 5 µM KY - 02061 in DMSO for 14 days . The cells were subjected to immunofluorescence analyses to visualize Runx2 ( green ) and β - catenin ( red ) . The cell nuclei were counterstained with DAPI ( blue ) .

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C. Primary calvaria cells were treated with DMSO or 5 µM KY-02061 in DMSO for 14 days. The cells were subjected to immunofluorescence analyses to visualize Runx2 (green) and β-catenin (red). The cell nuclei were counterstained with DAPI (blue).


D , E . Calvariae from 4 - day - old mice were cultured ex vivo for 7 days with KY - 02061 in DMSO ( D ) . The calvaria thicknesses were measured from the stained sections using Image Pro software ( E ) . [ n = 3 ]

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

D, E. Calvariae from 4-day-old mice were cultured ex vivo for 7 days with KY-02061 in DMSO (D). The calvaria thicknesses were measured from the stained sections using Image Pro software (E). [n=3]


B . A competition curve for the Dvl - CXXC5 interaction by KY - 02327 .

B. A competition curve for the Dvl-CXXC5 interaction by KY-02327.


C . Fluorescence quenching plot of Dvl PDZ domain upon addition of different amounts of KY - 02327 . From top to bottom final moral ratio between Dvl PDZ and KY - 02327 was , 1 : 0 , 1 : 1 , 1 : 2 , 1 : 3 , 1 : 4 , 1 : 5 , 1 : 6 , 1 : 7 , 1 : 8 , 1 : 9 , 1 : 10 , 1 : 15 , 1 : 20 and 1 : 25 , respectively .

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

C. Fluorescence quenching plot of Dvl PDZ domain upon addition of different amounts of KY-02327. From top to bottom final moral ratio between Dvl PDZ and KY-02327 was, 1:0, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20 and 1:25, respectively.


D - F . 1H - 15N - HSQC analyses were performed to analyze the interaction of 15N - labelled Dvl - PDZ domain with KY - 02327 . The 1H - 15N - HSQC spectrum of different molar ratios ( Dvl PDZ domain : KY - 02327 ) are displayed as red ( 1 : 0 ) , yellow ( 1 : 5 ) , green ( 1 : 10 ) , and magenta ( 1 : 20 ) ( D , residues with meaningful chemical shift change are indicated by arrows ) . Plot of chemical shift changes ( Δδ ) as a function of residue number in molecular ratio 1 : 20 ( E , a red - colored line indicates the line for Δδ = 0 . 05 ) . The residues with Δδ greater than 0 . 05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure ( F ) .

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

D-F. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02327. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02327) are displayed as red (1:0), yellow (1:5), green (1:10), and magenta (1:20) (D, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:20 (E, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (F).


G . Molecular docking of KY - 02061 or KY - 02327 to Dvl PDZ domain were analyzed by in silico experiments . The superimposed structure of KY - 02061 ( yellow ) and KY - 02327 ( cyan ) on the surface of Dvl PDZ domain ( gray ) was visualized .

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

G. Molecular docking of KY-02061 or KY-02327 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of KY-02061 (yellow) and KY-02327 (cyan) on the surface of Dvl PDZ domain (gray) was visualized.


A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV - β - gal . After 24 h , the cells were treated with indicated dose of KY - 02327 for 2 days . The luciferase activities of whole cell lysates were measured and normalized with β - galactosidase activities . [ n = 3 ]

O O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with indicated dose of KY-02327 for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]


B - C . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 2 days . β - Catenin and α - tubulin were detected by immunoblot .

O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O

B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot.


B - C . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 2 days . β - Catenin and α - tubulin were detected by immunoblot ( B ) or immunofluorescence ( C ) analyses . Nuclei were counterstained with DAPI ( C , blue ) .

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

B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot (B) or immunofluorescence (C) analyses. Nuclei were counterstained with DAPI (C, blue).


D . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 14 days . The mRNA level of collagen 1a ( Col1a ) was measured by quantitative real - time qRT - PCR . [ n = 3 ]

O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

D. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 14 days. The mRNA level of collagen 1a (Col1a) was measured by quantitative real-time qRT-PCR. [n=3]


E . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 21 days . The mRNA level of osteocalcin ( OCN ) was measured by qRT - PCR . [ n = 3 ]

O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

E. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 21 days. The mRNA level of osteocalcin (OCN) was measured by qRT-PCR. [n=3]


A - F . Vehicle or 20 mg of KY - 02327 per kg of animal body weight ( mpk ) was orally administered or 0 . 06 mpk of the N - terminal fragment of PTH ( amino acids 1 - 34 ) was subcutaneously injected into the Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 weeks [ n = 4 ] . After calcein injection , the prepared femoral sections of the OVX mice were examined under fluorescence microscope to visualize integrated calcein ( A , green , arrows ) . Nuclei were counterstained with DAPI ( A , blue ) . Osteoblast numbers ( B ) and calcein double labelled surfaces ( C ) were measured in the femoral sections .

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

A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. After calcein injection, the prepared femoral sections of the OVX mice were examined under fluorescence microscope to visualize integrated calcein (A, green, arrows). Nuclei were counterstained with DAPI (A, blue). Osteoblast numbers (B) and calcein double labelled surfaces (C) were measured in the femoral sections.


A - F . Vehicle or 20 mg of KY - 02327 per kg of animal body weight ( mpk ) was orally administered or 0 . 06 mpk of the N - terminal fragment of PTH ( amino acids 1 - 34 ) was subcutaneously injected into the Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 weeks [ n = 4 ] . The mouse femurs were analyzed using micro - CT and BMD ( D ) and BV / TV ( E ) were calculated from the micro - CT data .

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

A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The mouse femurs were analyzed using micro-CT and BMD (D) and BV/TV (E) were calculated from the micro-CT data.


A - F . Vehicle or 20 mg of KY - 02327 per kg of animal body weight ( mpk ) was orally administered or 0 . 06 mpk of the N - terminal fragment of PTH ( amino acids 1 - 34 ) was subcutaneously injected into the Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 weeks [ n = 4 ] . The three - dimensional images of femoral trabecular bone were reconstructed ( F ) , and trabecular number ( G ) and trabecular separation ( H ) were calculated .

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

A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The three-dimensional images of femoral trabecular bone were reconstructed (F), and trabecular number (G) and trabecular separation (H) were calculated.


B , C Kinetic change of mRNA expression of Top1 , Btbd1 , Tdp1 , and Srsf1 in microglia following LPS / IFNγ stimulation ( n = 6 , technical replicates ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Statistical analyses were performed using two - way ANOVA with Dunnett ' s Multiple Comparison test for ( B and C ) ,

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

B, C Kinetic change of mRNA expression of Top1, Btbd1, Tdp1, and Srsf1 in microglia following LPS/IFNγ stimulation (n = 6, technical replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using two-way ANOVA with Dunnett's Multiple Comparison test for (B and C),


D , E The protein expression of TOP1 was detected by Western blotting ( n = 3 , biological replicates ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

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

D, E The protein expression of TOP1 was detected by Western blotting (n = 3, biological replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


F - H Immunostaining of TOP1 ( red ) in stimulated microglia ( 4 h after LPS / IFNγ stimulation ) or resting microglia , co - stained with Hoechst ( blue ) ( bar = 20 μm ) ; for the lower - left panel in ( H ) , n = 3 technical replicates per group ( 2 to 5 random fields per well were analyzed ) ; for the lower right panel , 201 cells from unstimulated microglia and 256 cells from stimulated microglia were analyzed . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Immunostaining of TOP1 (red) in stimulated microglia (4 h after LPS/IFNγ stimulation) or resting microglia, co-stained with Hoechst (blue) (bar = 20 μm); for the lower-left panel in (H), n = 3 technical replicates per group (2 to 5 random fields per well were analyzed); for the lower right panel, 201 cells from unstimulated microglia and 256 cells from stimulated microglia were analyzed. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using the student's unpaired two-tailed t-test.


G Immunofluorescent staining of spinal cord sections from mice at EAE peak and control mice with anti - F4 / 80 ( green ) , TOP1 ( magenta ) , and nuclei with Hoechst ( blue ) . For the left panels , bar = 400 μm ; for the magnified panels , bar = 100 μm . The yellow arrowhead indicates a representative F4 / 80 + cell with high TOP1 expression .

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

G Immunofluorescent staining of spinal cord sections from mice at EAE peak and control mice with anti-F4/80 (green), TOP1 (magenta), and nuclei with Hoechst (blue). For the left panels, bar = 400 μm; for the magnified panels, bar = 100 μm. The yellow arrowhead indicates a representative F4/80+ cell with high TOP1 expression.


I A magnified image of the selected region ( white box ) in ( G ) ; bar = 50 F4 / 80 + cell with increased TOP1 expression in the nucleus in ( G ) . J The mean fluorescent intensity of TOP1 expression in the white matter in ( G ) was graphed ; n = 3 to 4 mice per group with 3 - 5 sections from each mouse . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

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

I A magnified image of the selected region (white box) in (G); bar = 50 F4/80+ cell with increased TOP1 expression in the nucleus in (G). J The mean fluorescent intensity of TOP1 expression in the white matter in (G) was graphed; n = 3 to 4 mice per group with 3-5 sections from each mouse. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


K TOP1 expression in the spinal cord homogenates of EAE mice dissected at disease peak ( scored 3 ) or control mice were examined by Western blotting . L The quantification of TOP1 protein expression of Western blot bands in ( K ) with normalization to GAPDH and β - actin ( n = 3 biological samples ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

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

K TOP1 expression in the spinal cord homogenates of EAE mice dissected at disease peak (scored 3) or control mice were examined by Western blotting. L The quantification of TOP1 protein expression of Western blot bands in (K) with normalization to GAPDH and β-actin (n = 3 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


M , Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h ( n = 4 or 5 biological samples ) .

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

M, Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples).


TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h ( n = 4 or 5 biological samples ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

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

TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


O Microglia were sorted from LPS challenged mice ( i . p ) after 4 h or naive mice , and the mRNA expression of TOP1 was graphed ( n = 5 or 6 biological samples ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O Microglia were sorted from LPS challenged mice (i.p) after 4 h or naive mice, and the mRNA expression of TOP1 was graphed (n = 5 or 6 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


P Brain sections from healthy control and MS brain tissues , including the active MS lesion , chronic active lesion , and the normal - appearing white matter ( NAWM ) , were stained with anti - HLA - DR , anti - TOP1 , and co - stained with Hoechst ( bar = 50 μm ) .

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

P Brain sections from healthy control and MS brain tissues, including the active MS lesion, chronic active lesion, and the normal-appearing white matter (NAWM), were stained with anti-HLA-DR, anti-TOP1, and co-stained with Hoechst (bar = 50 μm).


C , D Immunostaining of iNOS ( red ) and Phalloidin ( green ) in microglia ( n = 3 technical replicates with more than 5 random fields per well ) ( bar = 50 μm ) . Data information : Data shown are mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . = no statistical significance . Statistical analyses were performed using one - way ANOVA with Dunnett ' s Multiple Comparison Test for ( D )

O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D Immunostaining of iNOS (red) and Phalloidin (green) in microglia (n = 3 technical replicates with more than 5 random fields per well) (bar = 50 μm). Data information: Data shown are mean ± s.e.m. *P<0.05; **P<0.01; ***P<0.001; n.s. = no statistical significance. Statistical analyses were performed using one-way ANOVA with Dunnett's Multiple Comparison Test for (D)


B Microglia in the hippocampus and thalamus were stained with Iba1 ( red ) ; bar = 50 μm ; microglia in the yellow boxes were presented in ( E ) .

O B-CELL_TYPE O O B-TISSUE O B-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O

B Microglia in the hippocampus and thalamus were stained with Iba1 (red); bar = 50 μm; microglia in the yellow boxes were presented in (E).


C , D Fluorescent intensity of Iba1 in hippocampus and thalamus in ( B ) ; n = 2 , 4 , 5 mice for naive , LPS , and LPS + TPT group , respectively ( 2 - 6 sections per mouse were analyzed ) . Data information : Data shown are mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . = no statistical significance . Statistical analyses comparing the LPS _ PBS group with the LPS _ TPT group were performed using the Student ' s unpaired two - tailed t -

O O O O O O B-GENEPROD O B-TISSUE O B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

C, D Fluorescent intensity of Iba1 in hippocampus and thalamus in (B); n = 2, 4, 5 mice for naive, LPS, and LPS + TPT group, respectively (2-6 sections per mouse were analyzed). Data information: Data shown are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = no statistical significance. Statistical analyses comparing the LPS_PBS group with the LPS_TPT group were performed using the Student's unpaired two-tailed t-test


L , M Cytokine array on brain homogenates after 24 h of LPS challenge ( n = 2 mice for the naïve group ; n = 3 mice for the LPS and LPS + TPT groups ) .

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

L, M Cytokine array on brain homogenates after 24 h of LPS challenge (n = 2 mice for the naïve group; n = 3 mice for the LPS and LPS + TPT groups).


K Spinal cord sections dissected from Day 30 from the EAE batch in ( H ) were stained with CD11b ( red ) , Fluoromyelin ( green ) , and Hoechst ( blue ) ( bar = 200 μm ) . L The CD11b immunofluorescent intensity in ( K ) was graphed ( n = 2 mice for the control group ; n = 4 mice for the EAE - PBS and EAE - TPT groups ) . M The percentage of demyelinated area in the white matter of the spinal cord was graphed ( n = 2 mice for the control group , n = 4 mice for the EAE - PBS and EAE - TPT groups ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; the Student ' s unpaired two - tailed t - test was used for comparing the EAE - PBS group with the EAE - TPT group in L , and M .

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

K Spinal cord sections dissected from Day 30 from the EAE batch in (H) were stained with CD11b (red), Fluoromyelin (green), and Hoechst (blue) (bar = 200 μm). L The CD11b immunofluorescent intensity in (K) was graphed (n = 2 mice for the control group; n = 4 mice for the EAE-PBS and EAE-TPT groups). M The percentage of demyelinated area in the white matter of the spinal cord was graphed (n = 2 mice for the control group, n = 4 mice for the EAE-PBS and EAE-TPT groups). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; the Student's unpaired two-tailed t-test was used for comparing the EAE-PBS group with the EAE-TPT group in L, and M.


A RNA - seq . of freshly isolated , proliferating and differentiating satellite cells reveals an increased expression of linc - MYH in proliferating and differentiating MuSCs , but no expression is found in in freshly isolated MuSCs .

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

A RNA-seq. of freshly isolated, proliferating and differentiating satellite cells reveals an increased expression of linc-MYH in proliferating and differentiating MuSCs, but no expression is found in in freshly isolated MuSCs.


Expression of linc - MYH is confined to skeletal muscle in mouse ( B ) tissues .

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Expression of linc-MYH is confined to skeletal muscle in mouse (B) tissues.


Expression of linc - MYH is confined to skeletal muscle in human ( C ) tissues .

Expression of linc-MYH is confined to skeletal muscle in human (C) tissues.


D Expression of linc - MYH is not detected in limb buds at E10 . 5 ( n = 3 ) , E13 . 5 ( n = 3 ) and E15 . 5 ( n = 3 ) . Expression of linc - MYH in hindlimb muscle of E18 . 5 , newborn and 7 day old animals ( n = 3 / 3 / 3 ) . Expression of linc - MYH in m . soleus ( soleus ) , m . tibialis anterior ( TA ) and and m . extensor digitorum longus ( EDL ) muscle at 3 weeks ( n = 3 ) and 8 weeks of age ( n = 5 ) . Biological replicates were used for all PCR experiments , data are mean ± S . E . M .

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

D Expression of linc-MYH is not detected in limb buds at E10.5 (n = 3), E13.5 (n = 3) and E15.5 (n = 3). Expression of linc-MYH in hindlimb muscle of E18.5, newborn and 7 day old animals (n = 3/3/3). Expression of linc-MYH in m. soleus (soleus), m. tibialis anterior (TA) and and m. extensor digitorum longus (EDL) muscle at 3 weeks (n = 3) and 8 weeks of age (n = 5). Biological replicates were used for all PCR experiments, data are mean ± S.E.M.


E Murine linc - Myh is localized in nuclear extracts of C2C12 cells . Xist ( nuclear ) and Gapdh ( mainly cytoplasmic ) were used as controls ( n = 2 nuclear / 3 cytoplasmic biological replicates . ) Data are mean ± S . E . M .

O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-SUBCELLULAR O O B-CELL_LINE O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

E Murine linc-Myh is localized in nuclear extracts of C2C12 cells. Xist (nuclear) and Gapdh (mainly cytoplasmic) were used as controls (n = 2 nuclear / 3 cytoplasmic biological replicates.) Data are mean ± S.E.M.


F - H RNA - FISH identifies linc - Myh in nuclei ( DAPI , blue ) of C2C12 myoblasts . ( G ) Adipor ( cytoplasmic ) and ( H ) Xist ( nuclear ) were used as controls .

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

F-H RNA-FISH identifies linc-Myh in nuclei (DAPI, blue) of C2C12 myoblasts. (G) Adipor (cytoplasmic) and (H) Xist (nuclear) were used as controls.


( J ) Heat map of proteins pulled down in the same experiments as in I , demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc - MYH . Blue color indicates pull - down of the respective protein , white color indicates that the protein was not detected in the respective sample .

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

(J) Heat map of proteins pulled down in the same experiments as in I, demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc-MYH. Blue color indicates pull-down of the respective protein, white color indicates that the protein was not detected in the respective sample.


RNA - IP experiments using an anti - INO80 antibody to isolate the INO80 complex from murine C2C12 cells ( K ) INO80 in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-ORGANISM B-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from murine C2C12 cells (K) INO80 in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.


RNA - IP experiments using an anti - INO80 antibody to isolate the INO80 complex from human HSMM cells ( L ) . INO80 II in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR .

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RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from human HSMM cells (L). INO80 II in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.


Body weight ( A ) of male linc - MYH KO and ctrl mice at 10 weeks ( n = 24 KO / 17 WT ; Student ' s t test , two - tailed , * * p < 0 . 01 )

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Body weight (A) of male linc-MYH KO and ctrl mice at 10 weeks (n = 24 KO/17 WT; Student's t test, two-tailed, **p < 0.01)


tibia length ( B ) of male linc - MYH KO and ctrl mice of 10 weeks ( n = 9 KO / 13 WT ; Student ' s t test , two - tailed , not significant ) . Data are mean ± S . E . M .

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

tibia length (B) of male linc-MYH KO and ctrl mice of 10 weeks (n = 9 KO/13 WT; Student's t test, two-tailed, not significant). Data are mean ± S.E.M.


C , D Weight of m . tibialis anterior ( TA ) and m . digitorum longus ( EDL ) of male linc - MYH KO and ctrl mice at 10 weeks ( n = 9 KO / 10 WT for TA , n = 10 KO / 11 WT for EDL ; Student ' s t test , two - tailed , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data are mean ± S . E . M .

O O O B-EXP_ASSAY O O O B-TISSUE I-TISSUE O B-TISSUE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O 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, D Weight of m. tibialis anterior (TA) and m. digitorum longus (EDL) of male linc-MYH KO and ctrl mice at 10 weeks (n = 9 KO/10 WT for TA, n = 10 KO/11 WT for EDL; Student's t test, two-tailed, **p < 0.01, ***p<0.001). Data are mean ± S.E.M.


Size distribution of fibers in cross sections of TA ( E , F ) and EDL ( G , H ) muscle in male linc - MYH KO ( blue ) and ctrl ( grey ) animals at 10 weeks . A significant increase of fiber size was observed for TA and EDL muscles of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 fibers per animal were counted ) . Data are mean ± S . E . M .

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

Size distribution of fibers in cross sections of TA (E, F) and EDL (G, H) muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.


Size distribution of fibers in cross sections of TA and EDL muscle in male linc - MYH KO ( blue ) and ctrl ( grey ) animals at 10 weeks . A significant increase of fiber size was observed for TA and EDL muscles of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 fibers per animal were counted ) . Data are mean ± S . E . M .

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

Size distribution of fibers in cross sections of TA and EDL muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.


K Numbers of myonuclei in cross sections of TA and EDL muscle ( TA : n = 6 KO / 6 WT , EDL n = 3 KO / 3 WT animals , Mann - Whitney test one - tailed , * * p < 0 . 01 , * p = 0 . 05 ) Data are mean ± S . E . M .

O O O B-SUBCELLULAR O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Numbers of myonuclei in cross sections of TA and EDL muscle (TA: n = 6 KO/6 WT, EDL n = 3 KO/3 WT animals, Mann-Whitney test one-tailed, **p<0.01, *p = 0.05) Data are mean ± S.E.M.


L - O Number of myonuclei / fiber and of Pax7pos ( red ) MuSCs ( red arrows ) on myofibers isolated from flexor digitorum brevis muscle . Nuclei were stained using DAPI ( blue ) . Scale bar in L indicates 50 µm for L and M . ( N ) The number of myonuclei / isolated fiber is increased in linc - MYH KO compared to WT myofibers ( n = 4 KO / 4 WT , > 26 fibers / animal ; Mann - Whitney test two - tailed , * p < 0 . 05 ) . ( O ) Number of MuSCs on isolated fibers from linc - MYH KO and WT mice ( n = 4 KO / 4 WT animals , Mann - Whitney test two - tailed ; * * * * p < 0 . 0001 ) . Data are mean ± S . E . M .

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

L-O Number of myonuclei / fiber and of Pax7pos (red) MuSCs (red arrows) on myofibers isolated from flexor digitorum brevis muscle. Nuclei were stained using DAPI (blue). Scale bar in L indicates 50 µm for L and M. (N) The number of myonuclei / isolated fiber is increased in linc-MYH KO compared to WT myofibers (n = 4 KO/4 WT, > 26 fibers/animal; Mann-Whitney test two-tailed, * p < 0.05). (O) Number of MuSCs on isolated fibers from linc-MYH KO and WT mice (n = 4 KO/4 WT animals, Mann-Whitney test two-tailed; ****p < 0.0001). Data are mean ± S.E.M.


P - S Number of Pax7 ( red ) positive MuSCs in cross sections of tibialis anterior muscle ( P - R ) ; the relative amount of quiescent MuSCs identified by double staining ( yellow arrows ) for Pax7 and CalcR ( green ) does not significantly differ between WT and linc - MYH KO animals ( S ) . Nuclei were stained with DAPI ( blue ) . Scale bar in P indicates 25 µm for P and Q . ( n = 3 KO / 3 WT animals , Mann - Whitney test one - tailed ; * p = 0 . 05 , ns : not significant ) . All data are mean ± S . E . M .

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

P-S Number of Pax7 (red) positive MuSCs in cross sections of tibialis anterior muscle (P-R); the relative amount of quiescent MuSCs identified by double staining (yellow arrows) for Pax7 and CalcR (green) does not significantly differ between WT and linc-MYH KO animals (S). Nuclei were stained with DAPI (blue). Scale bar in P indicates 25 µm for P and Q. (n = 3 KO/3 WT animals, Mann-Whitney test one-tailed; *p = 0.05, ns: not significant). All data are mean ± S.E.M.


T The number of MuSC - related events in FACS experiments is significantly increased in linc - MYH - KO animals ( n = 7 KO / 7 WT , Student ' s t test , two - tailed , * p = 0 . 0126 ) . Data are mean ± S . E . M .

O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

T The number of MuSC-related events in FACS experiments is significantly increased in linc-MYH-KO animals (n = 7 KO/7 WT, Student's t test, two-tailed, *p = 0.0126). Data are mean ± S.E.M.


U - V Structure of MuSCs and the heterochromatin content of MuSC nuclei is not changed in linc - MYH KO ( V ) compared to WT mice ( U ) .

O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O B-CELL_TYPE B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O O

U-V Structure of MuSCs and the heterochromatin content of MuSC nuclei is not changed in linc-MYH KO (V) compared to WT mice (U).


RNA in situ hybridization proximity ligation assay ( rISH - PLA ) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al . . ( B - C ) rISH - PLA confirms the proximity of linc - MYH to endogenous INO80 - V5 in the nucleus of proliferating WT MuSCs ( B ) . The specific signal is lost in linc - MYH deficient MuSCs ( C ) . ( n = 3 WT / 3 KO biological replicates ; Mann - Whitney test , * p = 0 . 05 ) . Data are mean ± S . E . M .

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

RNA in situ hybridization proximity ligation assay (rISH-PLA) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al.. (B-C) rISH-PLA confirms the proximity of linc-MYH to endogenous INO80-V5 in the nucleus of proliferating WT MuSCs (B). The specific signal is lost in linc-MYH deficient MuSCs (C). (n = 3 WT/3 KO biological replicates; Mann-Whitney test, *p = 0.05). Data are mean ± S.E.M.


E - I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 ( green ) and EdU incorporation ( red ) . MuSCs without ( green arrows ) and with EdU - labeling ( red arrows ) are indicated . ( E , F ) Deletion of linc - MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU - positive MuSCs . ( G - I ) The increase in both the number of MuSCs and EdU positive MuSCs in linc - MYH mutant muscle is abolished by constitutive ( G : Pax7 - Crepos / INO80 - / - , Ino80 KO ; H : linc - MYH - / - / Pax7 - Crepos / INO80 - / - , dKO ) and induced ( I : linc - MYH - / - / Pax7 - CreERT2pos / INO80 - / - , dKO CreERT ) deletion of INO80 in Pax7 expressing cells .

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

E-I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 (green) and EdU incorporation (red). MuSCs without (green arrows) and with EdU-labeling (red arrows) are indicated. (E, F) Deletion of linc-MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU-positive MuSCs. (G-I) The increase in both the number of MuSCs and EdU positive MuSCs in linc-MYH mutant muscle is abolished by constitutive (G: Pax7-Crepos / INO80-/-, Ino80 KO; H: linc-MYH-/- / Pax7-Crepos / INO80-/-, dKO) and induced (I: linc-MYH-/- / Pax7-CreERT2pos / INO80-/-, dKO CreERT) deletion of INO80 in Pax7 expressing cells.


Statistical evaluation of the number of muscle stem cells observed in TA muscles ( J ) . ; ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

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

Statistical evaluation of the number of muscle stem cells observed in TA muscles (J). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


Statistical evaluation of the ratio of EdU - incorporating MuSCs relative to all Pax7 - positive MuSCs ( K ) . ; ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-CELL_TYPE O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Statistical evaluation of the ratio of EdU-incorporating MuSCs relative to all Pax7-positive MuSCs (K). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes ( L ) ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

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

Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes (L) (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


A - D Isolated linc - MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates . Deletion of INO80 eliminates the difference between Pax7 - Crepos / INO80 - / - ( Ino80 KO ) and linc - MYH - / - / linc - MYH - / - / Pax7 - Crepos / INO80 - / - ( dKO ) . Images are representative images for cultures after 13 days in culture .

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

A-D Isolated linc-MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates. Deletion of INO80 eliminates the difference between Pax7-Crepos / INO80-/- (Ino80 KO) and linc-MYH-/- / linc-MYH-/-/ Pax7-Crepos / INO80-/- (dKO). Images are representative images for cultures after 13 days in culture.


E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs . Cell confluence was monitored by a live cell imaging analysis system for 13 days ( n = 7 WT / 6 linc - MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs ) .

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

E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs. Cell confluence was monitored by a live cell imaging analysis system for 13 days (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs).


F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation ( n = 7 WT / 6 linc - MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs , Student ' s t test , two - tailed ; * p < 0 . 05 , ns = not significant ) . Data are mean ± S . E . M .

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F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs, Student's t test, two-tailed; *p < 0.05, ns = not significant). Data are mean ± S.E.M.


G - I Gene set enrichment analysis ( GSEA , broadinstitute . org ) of microarray data from proliferating MuSCs . Comparison of linc - MYH - / - vs . WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation ( G ) , enrichment of genes containing cis regulatory elements for YY1 ( H ) and p53 ( I ) . Enrichments are highly significant using the most conservative familywise - error rate method ( FWER ) . Enrichments were lost in linc - MYH - / - MuSCs after additional deletion of INO80 ( dKOs ) compared to Pax7 - Crepos / INO80 - / - MuSCs .

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

G-I Gene set enrichment analysis (GSEA, broadinstitute.org) of microarray data from proliferating MuSCs. Comparison of linc-MYH-/- vs. WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation (G), enrichment of genes containing cis regulatory elements for YY1 (H) and p53 (I). Enrichments are highly significant using the most conservative familywise-error rate method (FWER). Enrichments were lost in linc-MYH-/- MuSCs after additional deletion of INO80 (dKOs) compared to Pax7-Crepos / INO80-/- MuSCs.


Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G - I in linc - MYH - / - vs . WT MuSCs and in linc - MYH - / - / Pax7 - Crepos / INO80 - / - dKO vs . Pax7 - Crepos / INO80 - / - MuSCs . Expression of up - regulated genes in linc - MYH - / - was normalized after additional deletion of INO80 ( dKOs ) , indicating that effects of linc - MYH completely depend on INO80 ( WT n = 4 , linc - MYH - / - n = 4 , Pax7 - Crepos / INO80 - / - n = 4 , Pax7 - Cre dKO n = 3 animals used for independent isolations of MuSCs ) .

O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O B-CELL_TYPE O O

Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs).


A - D γ - H2AX staining of proliferating MuSCs from WT ( A ) , linc - MYH deficient ( B ) , Pax7 - Crepos / INO80 KO ( C ) , and linc - MYH / INO80 dKO ( D ) mice . Deletion of INO80 results in increased γ - H2AX signals while deletion of linc - MYH has no effects ( asterisk : low γ - H2AX signals , arrow : γ - H2AX - foci detected in nuclei , arrowhead : pan - nuclear γ - H2AX signal .

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

A-D γ-H2AX staining of proliferating MuSCs from WT (A), linc-MYH deficient (B), Pax7-Crepos / INO80 KO (C), and linc-MYH/INO80 dKO (D) mice. Deletion of INO80 results in increased γ-H2AX signals while deletion of linc-MYH has no effects (asterisk: low γ-H2AX signals, arrow: γ-H2AX-foci detected in nuclei, arrowhead: pan-nuclear γ-H2AX signal.


E - G Statistical evaluation of the presence of γ - H2AX signals in MuSCs of different genotypes ( n = 3 / 3 / 3 / 3 animals used for independent isolations of MuSCs , two technical replicates for each animal and at least 164 cells were counted , Mann - Whitney test , * * p < 0 . 01 . Data are mean ± s . e . m . ) .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O B-ORGANISM O O O O O B-CELL_TYPE O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-G Statistical evaluation of the presence of γ-H2AX signals in MuSCs of different genotypes (n = 3/3/3/3 animals used for independent isolations of MuSCs, two technical replicates for each animal and at least 164 cells were counted, Mann-Whitney test, **p<0.01. Data are mean ± s.e.m.).


H - I Gene set enrichment analysis of microarray data obtained for proliferating MuSCs shows enrichment of genes related to activity of ATR in response to replication stress recovery ( H ) and enrichment of genes involved in chromosome maintenance ( I ) in WT compared to Pax7 - Crepos INO80 - / - MuSCs . Enrichment of these or similar gene sets was not observed when comparing WT to linc - MYH - / - MuSCs .

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

H-I Gene set enrichment analysis of microarray data obtained for proliferating MuSCs shows enrichment of genes related to activity of ATR in response to replication stress recovery (H) and enrichment of genes involved in chromosome maintenance (I) in WT compared to Pax7-Crepos INO80-/- MuSCs. Enrichment of these or similar gene sets was not observed when comparing WT to linc-MYH-/- MuSCs.


A , B Co - Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc - MYH KO mice followed by Western Blot analysis . ( B ) Statistical analysis of Co - IP / Western Blot data . The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc - MYH , while the amounts of YY1 , WDR5 and TFPT pulled down by INO80 increase ( n = 3 WT / 3 linc - MYH KO animals ; one - way ANOVA , multiple comparisons by Fishers LSD test , * p < 0 . 05 ) . Data are mean ± S . E . M .

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

A, B Co-Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc-MYH KO mice followed by Western Blot analysis. (B) Statistical analysis of Co-IP/Western Blot data. The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc-MYH, while the amounts of YY1, WDR5 and TFPT pulled down by INO80 increase (n = 3 WT / 3 linc-MYH KO animals; one-way ANOVA, multiple comparisons by Fishers LSD test, *p<0.05). Data are mean ± S.E.M.


C - K Proximity ligation assays between INO80 and YY1 ( C , D ) , INO80 and WDR5 ( F , G ) , and INO80 and RuvBl2 ( I , J ) in proliferating MuSCs . Red signals indicate close proximity of INO80 - V5 and the respective interacting protein . Nuclei were stained using DAPI ( blue ) . Statistical evaluation of proximity ligation assays indicating increased interactions of INO80 - V5 and YY1 ( E , n = 3 / 3 animals , 3 independent wells per animal , two - tailed students t - test , * p < 0 . 05 ) and of INO80 - V5 to WDR5 ( H , n = 2 ( WT ) / 2 ( KO ) animals , 3 independent wells per animal , two - tailed students t - test , * * p < 0 . 01 ) . No increase in proximity was detected between Ino80 - V5 and Ruvbl2 ( K , n = 2 ( WT ) / 2 ( KO ) animals , 3 independent wells per animal , two - tailed students t - test , ns : not significant ) . Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays . No signals were detected for these controls . Data are mean ± S . E . M .

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

C-K Proximity ligation assays between INO80 and YY1 (C, D), INO80 and WDR5 (F, G), and INO80 and RuvBl2 (I, J) in proliferating MuSCs. Red signals indicate close proximity of INO80-V5 and the respective interacting protein. Nuclei were stained using DAPI (blue). Statistical evaluation of proximity ligation assays indicating increased interactions of INO80-V5 and YY1 (E, n = 3/3 animals, 3 independent wells per animal, two-tailed students t-test, *p<0.05) and of INO80-V5 to WDR5 (H, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, **p<0.01). No increase in proximity was detected between Ino80-V5 and Ruvbl2 (K, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, ns: not significant). Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays. No signals were detected for these controls. Data are mean ± S.E.M.


( A ) Coomassie stained SDS - PAGE showing purified T . thermophila tubulin .

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(A) Coomassie stained SDS-PAGE showing purified T. thermophila tubulin.


( B ) A representative cryo - electron micrograph of paclitaxel - stabilized T . thermophila MTs .

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(B) A representative cryo-electron micrograph of paclitaxel-stabilized T. thermophila MTs.


( C ) Kymographs obtained by TIRF microscopy showing the dynamics of T . thermophila MTs at the indicated concentrations in µM .

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(C) Kymographs obtained by TIRF microscopy showing the dynamics of T. thermophila MTs at the indicated concentrations in µM.


( D ) Plot showing the time course of T . thermophila cell growth in the presence of CA4 ( 20µM ) or parabulin ( 50µM ; error bars corresponding to the standard deviation of triplicate biological measurements ) . Control experiments are done with DMSO alone .

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(D) Plot showing the time course of T. thermophila cell growth in the presence of CA4 (20µM) or parabulin (50µM; error bars corresponding to the standard deviation of triplicate biological measurements). Control experiments are done with DMSO alone.


( A ) Plot showing quantification of the T . thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in the presence of the indicated drugs . Error bars represent the mean + / - 95 % CI . For each concentration , data is obtained from at least 2 independent experiments . For growth rates and growth lengths , n = 41 ( DMSO ) , n = 4 ( 0 . 04uM parabulin ) , n = 34 ( 0 . 04 uM colchicine ) . Rates were compared using one - way ANOVA with Tukey ' s multiple comparison test ; DMSO vs parabulin P < 0001 ( * * * * ) ; DMSO vs colchicine p = 0 . 40 ( n . s . , not significant ) . ( B ) T . thermophila MT growth rates in the presence of the indicated drugs ( 40 μM ) . Error bars represent the mean + / - 95 % CI ( n = 4 ) . Rates were compared using one - way ANOVA with Tukey ' s multiple comparison test ; DMSO vs parabulin P = 0 . 25 ; DMSO vs colchicine P = 0 . 20 ( n . s . , not significant ) .

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

(A) Plot showing quantification of the T. thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in the presence of the indicated drugs. Error bars represent the mean +/- 95% CI. For each concentration, data is obtained from at least 2 independent experiments. For growth rates and growth lengths, n=41(DMSO), n= 4 (0.04uM parabulin), n= 34 (0.04 uM colchicine). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P<0001 (****); DMSO vs colchicine p = 0.40 (n.s., not significant). (B) T. thermophila MT growth rates in the presence of the indicated drugs (40 μM). Error bars represent the mean +/- 95% CI (n=4). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P = 0.25; DMSO vs colchicine P = 0.20 (n.s., not significant).


( D ) Representative kymographs obtained by TIRF microscopy showing the effect of parabulin and colchicine on dynamic Tt - MTs .

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

(D) Representative kymographs obtained by TIRF microscopy showing the effect of parabulin and colchicine on dynamic Tt-MTs.


( A ) Evaluation of vertebrate cell toxicity using the MTT assay which detects metabolic activity proportionate to the number of viable cells in a sample . The absorbance measurements for all treatment conditions were normalized to the null control value which is set to 100 % growth . Sub - confluent human fibroblasts were used to visualize potential compound effects during proliferation . The results represent the average of 9 readings ( 3 biological replicates with 3 technical replicates of each treatment ) ± standard error of the mean .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Evaluation of vertebrate cell toxicity using the MTT assay which detects metabolic activity proportionate to the number of viable cells in a sample. The absorbance measurements for all treatment conditions were normalized to the null control value which is set to 100% growth. Sub-confluent human fibroblasts were used to visualize potential compound effects during proliferation. The results represent the average of 9 readings (3 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.


( B ) Replication of GFP - expressing T . gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging . Since vacuoles containing more than eight tachyzoites are not always easy to score , we tallied vacuoles above eight as being 8 + in size . At 36 hours , both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites ( three or more doublings ) . Vacuoles in parabulin - treated cultures contain significantly fewer parasites , indicating that replication is slowed . The graph shows three biological replicates with values normalized to 100 % for each condition and each replicate , error bars represent standard error of the mean between experiments .

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

(B) Replication of GFP-expressing T. gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging. Since vacuoles containing more than eight tachyzoites are not always easy to score, we tallied vacuoles above eight as being 8+ in size. At 36 hours, both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites (three or more doublings). Vacuoles in parabulin-treated cultures contain significantly fewer parasites, indicating that replication is slowed. The graph shows three biological replicates with values normalized to 100% for each condition and each replicate, error bars represent standard error of the mean between experiments.


( C ) The parabulin EC50 value for T . gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions . Error bars represent standard error of the mean . The results represent the average of 18 readings ( 6 biological replicates with 3 technical replicates of each treatment ) ± standard error of the mean .

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

(C) The parabulin EC50 value for T. gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions. Error bars represent standard error of the mean. The results represent the average of 18 readings (6 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.


( D ) Phase contrast of extracellular parasite cultures . Serial passage of T . gondii in 40 - 80 μM parabulin induces accumulation of aberrant tachyzoite forms . These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding .

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

(D) Phase contrast of extracellular parasite cultures. Serial passage of T. gondii in 40-80 μM parabulin induces accumulation of aberrant tachyzoite forms. These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding.


A ) Solid phase transfection of nontargeting ( scrambled ) or PLK1 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 24 , 48 and 72 hours and imaged after DNA staining with Hoechst . Green arrowheads indicate examples of prometaphase arrested cells , and the red arrowheads indicate examples of dead cells due to Plk1 downregulation . Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments . Scale bar , 20 μm .

O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 24, 48 and 72 hours and imaged after DNA staining with Hoechst. Green arrowheads indicate examples of prometaphase arrested cells, and the red arrowheads indicate examples of dead cells due to Plk1 downregulation. Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments. Scale bar, 20 μm.


B ) Solid phase transfection of nontargeting ( scrambled ) or CCNA2 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours and imaged after DNA staining with Hoechst . C ) Quantification of nuclear size measurements from Fig 2B . Data derived from 3 independent experiments . P value ( scrambled vs CCNA2 ) < 2e - 16 , Kolmogorov - Smirnov test .

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

B) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. C) Quantification of nuclear size measurements from Fig 2B. Data derived from 3 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.


D ) Solid phase transfection of nontargeting ( scrambled ) , GOLGA2 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Golga2 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . E ) Quantification of experiments in Fig 2D . Data derived from 3 independent experiments . P value ( scrambled vs GOLGA2 ) < 2e - 16 , Mann - Whitney U test .

O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

D) Solid phase transfection of nontargeting (scrambled), GOLGA2 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Golga2 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. E) Quantification of experiments in Fig 2D. Data derived from 3 independent experiments. P value (scrambled vs GOLGA2) < 2e-16, Mann-Whitney U test.


F ) Solid phase transfection of nontargeting ( scrambled ) , MKI67 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Ki67 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . G ) Quantification of experiments in Fig 2F . Data derived from 3 independent experiments . P value ( scrambled vs MKI67 ) < 2e - 16 , Mann - Whitney U test .

F) Solid phase transfection of nontargeting (scrambled), MKI67 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. G) Quantification of experiments in Fig 2F. Data derived from 3 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test.