Elevation of mutant KLF4 shows same effect as WT KLF4 to Olaparib in BRCA1 mutant lines MDA - MB - 436 . No significant difference between expression of wild - type KLF4 KLF4 PARylation - deficient mutant was observed in MDA - MB - 436 .

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Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines MDA-MB-436 cells. No significant difference between expression of wild-type KLF4 and KLF4 PARylation-deficient mutant was observed in MDA-MB-436 cells.


Modulating KLF4 by knockdown or overexpression affects response of MDA - MB - 231 to Olaparib in clonogenic .

Modulating KLF4 by knockdown or overexpression affects cellular response of MDA-MB-231 to Olaparib in clonogenic assay.


Modulating KLF4 by knockdown or overexpression affects response of MCF7 to Olaparib in clonogenic .

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Modulating KLF4 by knockdown or overexpression affects cellular response of MCF7 to Olaparib in clonogenic assay.


Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib / Doxorubicin in MDA - MB - 231 ( Q ) Data are mean ± ; one - way ANOVA was used for statistical . asterisk in panels represents significant difference ( p < 0 . 05 ) . exact p - Values were supplied in Appendix Table S4 .

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Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib/Doxorubicin in MDA-MB-231(Q) Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. The asterisk in the panels represents the significant difference (p<0.05). The exact p-Values were supplied in Appendix Table S4.


Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib / Cisplatin in MCF7 Data are mean ± ; one - way ANOVA was used for statistical . asterisk in panels represents significant difference ( p < 0 . 05 ) . exact p - Values were supplied in Appendix Table S4 .

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Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib/Cisplatin in MCF7 Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. The asterisk in the panels represents the significant difference (p<0.05). The exact p-Values were supplied in Appendix Table S4.


( A ) Depletion of KLF4 significantly enhances efficacy of Olaparib in killing TNBC ( 4T1 ) . 4T1 with stable expression of shKLF4 were implanted into mammary fat pad of BALB / c Nude mice . Drug treatment was started at 10th day . Placebo or Olaparib at dose of 100 mg / kg was administrated daily for twelve days . volumes were measured every other day . volumes were measured every other day . n = 9 per group , p values were labeled in figure , one - way ANOVA .

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(A) Depletion of KLF4 significantly enhances the efficacy of Olaparib in killing TNBC cells (4T1). 4T1 cells with stable expression of shKLF4 were implanted into mammary fat pad of BALB/c Nude mice. Drug treatment was started at 10th day. Placebo or Olaparib at the dose of 100 mg/kg was administrated daily for twelve days. Tumor volumes were measured every other day. Tumor volumes were measured every other day. n=9 per group, p values were labeled in figure, one-way ANOVA assay.


( B & C ) image of 4T1 xenograft tumors ( B ) tumors were weighed summarized ( C ) . n = 9 per group , p values were labeled in figure , one - way ANOVA .

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(B & C) The image of 4T1 xenograft tumors (B) and tumors were weighed and summarized (C). n=9 per group, p values were labeled in figure, one-way ANOVA assay.


combination effect when combine of Olaparib with ABT - 263 dasatinib in suppression MDA - MB - 231 xenograft growth . 8 SCID / Beige mice were injected 8x106 MDA - MB - 231 in matrigel ( 1 : 1 volume ) . When reach to 50mm3 , 100mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) was administrated by oral gavage daily , 30mg / kg ABT - 263 ( dissolve in 5 % DMSO ) was administrated by intraperitoneal injection every other day , 50mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) or placebo ( 2 % DMSO + 30 % PEG400 + saline ) were administrated by oral gavage every other day . Mice were treated with single or combination drugs for four . volumes were measured every other day ( F ) . image of MDA - MB - 231 xenograft tumors

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

The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (F). The image of MDA-MB-231 xenograft tumors


combination effect when combine of Olaparib with ABT - 263 dasatinib in suppression MDA - MB - 231 xenograft growth . 8 SCID / Beige mice were injected 8x106 MDA - MB - 231 in matrigel ( 1 : 1 volume ) . When reach to 50mm3 , 100mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) was administrated by oral gavage daily , 30mg / kg ABT - 263 ( dissolve in 5 % DMSO ) was administrated by intraperitoneal injection every other day , 50mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) or placebo ( 2 % DMSO + 30 % PEG400 + saline ) were administrated by oral gavage every other day . Mice were treated with single or combination drugs for four . volumes were measured every other day ( F ) . image of MDA - MB - 231 xenograft

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


combination effect when combine of Olaparib with ABT - 263 dasatinib in suppression MDA - MB - 231 xenograft growth . 8 SCID / Beige mice were injected 8x106 MDA - MB - 231 in matrigel ( 1 : 1 volume ) . When reach to 50mm3 , 100mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) was administrated by oral gavage daily , 30mg / kg ABT - 263 ( dissolve in 5 % DMSO ) was administrated by intraperitoneal injection every other day , 50mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) or placebo ( 2 % DMSO + 30 % PEG400 + saline ) were administrated by oral gavage every other day . Mice were treated with single or combination drugs for four . volumes were measured every other day ( G ) . tumors were weighed summarized ( H ) . Placebo ( n = 7 ) , ABT263 ( n = 9 ) , Dasatinib ( n = 8 ) , Olaparib ( n = 6 ) , Olaparib + ABT263 ( n = 9 ) , Olaparib + Dasatinib ( n = 10 ) , p values were labeled in figure , one - way ANOVA .

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The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (G). The tumors were weighed and summarized (H). Placebo (n=7), ABT263 (n=9), Dasatinib (n=8), Olaparib (n=6), Olaparib+ABT263 (n=9), Olaparib+Dasatinib (n=10), p values were labeled in figure, one-way ANOVA assay.


( I ) of H & E , KLF4 , PARP1 , p21 Bax in MDA - MB - 231 xenograft tumors . Scale bars , 100 μm . Combination of ABT - 263 or dasatinib with Olaparib treatment decrease KLF4 p21expression , but increase Bax expression in xenograft tumors .

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

(I) Staining of H & E, KLF4, PARP1, p21 and Bax in MDA-MB-231 xenograft tumors. Scale bars, 100 μm. Combination of ABT-263 or dasatinib with Olaparib treatment decrease KLF4 and p21expression, but increase Bax expression in xenograft tumors.


Kaplan - Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9 - hSynI - mWwox [ total n = 18 , spontaneously dead n = 6 , mice taken out for electrophysiology / electron microscopy / , are shown in yellow , n = 12 ] compared to mice injected with AAV9 - hSynI - GFP ( n = 6 ) or non - injected ( n = 8 ( P < 0 . 0001 , Log - rank Mantel - Cox ) .

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Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hSynI-mWwox [total n=18, spontaneously dead n=6, mice taken out for electrophysiology/electron microscopy/analysis, are shown in yellow, n=12] compared to mice injected with AAV9-hSynI-GFP (n=6) or the non-injected (n=8 (P <0.0001, Log-rank Mantel-Cox test).


Immunofluorescence showing expression of WWOX ( red ) in brain tissue ( cortex ) at P17 from WT Wwox null mice that were injected with AAV9 - hSynI - mWwox ( 2x1010 ) . Neurons are labelled with anti - NeuN antibody ( green ) . square box shows magnified . arrows show transgene expression in NeuN positive . Graph showing of NeuN WWOX double positive in different parts of brain ( Cortex , Hippocampus Cerebellum ) from KO mice injected with AAV - mWwox at P17 . NeuN WWOX double positive were calculated from 3 identical sagittal sections of AAV - mWwox injected mice ( n = 3 ) brains .

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Immunofluorescence images showing the expression of WWOX (red) in brain tissue (cortex) at P17 from WT and Wwox null mice that were injected with AAV9-hSynI-mWwox (2x1010). Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area. White arrows show the transgene expression in NeuN positive cells. Graph showing the percentage of NeuN and WWOX double positive cells in different parts of the brain (Cortex, Hippocampus and Cerebellum) from KO mice injected with AAV-mWwox at P17. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-mWwox injected mice (n=3) brains.


Kaplan - Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9 - hWWOX [ total n = 16 , alive n = 6 , spontaneously dead n = 6 , 4 mice ( shown in yellow ) were taken out for ) compared to non - injected ( n = 8 ) ] ( P < 0 . 0001 , Log - rank Mantel - Cox ) .

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Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hWWOX [total n=16, alive n=6, spontaneously dead n=6, 4 mice (shown in yellow) were taken out for analysis) compared to the non-injected (n=8)] (P <0.0001, Log-rank Mantel-Cox test).


D - F . Immunofluorescence showing expression of WWOX ( magenta ) in cortex ( D ) , Hippocampus ( E ) Cerebellum ( F ) at P19 from WT Wwox null mice that were injected with AAV - hWWOX ( 2x1010 ) or Wwox null alone . Neurons are labelled with anti - NeuN antibody ( green ) . square box shows magnified . Graph represents of neurons showing transgene ( NeuN WWOX positive ) expression at different parts ( cortex , hippocampus cerebellum ) of KO brain at P19 after AAV injection . NeuN WWOX double positive were calculated from 3 identical sagittal sections of AAV - hWWOX injected mice ( n = 3 ) brains .

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

D-F. Immunofluorescence images showing the expression of WWOX (magenta) in cortex (D), Hippocampus (E) and Cerebellum (F) at P19 from WT and Wwox null mice that were injected with AAV-hWWOX (2x1010) or Wwox null alone. Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area.   Graph represents the percentage of neurons showing the transgene (NeuN and WWOX positive) expression at different parts (cortex, hippocampus and cerebellum) of the KO brain at P19 after AAV injection. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-hWWOX injected mice (n=3) brains.


Representative traces of - attached recordings performed in WT ( blue ) , KO ( red ) KO injected with either with AAV9 - hSynI - mWwox ( KO + A - mW shown in purple ) or AAV9 - hSynI - hWWOX ( KO + A - hW shown in green ) pups at P18 - 21 days . Traces represent spontaneous neocortical activity ( action potentials shown ) . panels represent 12 s recording with an inset presenting a zoom - in of an 0 . 5 s interval . graph shows recordings ( P18 - 21 ) averages over 30 neurons from WT ( n = 3 ) , 30 neurons from KO + A - mWwox ( n = 3 ) , 30 neurons from KO + A - hWWOX ( n = 3 ) , 45 neurons from KO ( n = 3 ) .

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

Representative traces of cell-attached recordings performed in WT (blue), KO (red) and KO injected with either with AAV9-hSynI-mWwox (KO+A-mW shown in purple) or AAV9-hSynI-hWWOX (KO+A-hW shown in green) pups at P18-21 days. Traces represent spontaneous neocortical activity (action potentials shown). The panels represent 12 s recording with an inset presenting a zoom-in of an 0.5 s interval.   The graph shows the recordings (P18-21) averages over 30 neurons from WT (n=3), 30 neurons from KO+A-mWwox (n=3), 30 neurons from KO+A-hWWOX (n=3), and 45 neurons from KO (n=3).


Immunofluorescence with anti - GFAP ( shown in green ) anti - Iba1 ( shown in magenta ) antibodies showing expression of astrocytes microglia at P19 from WT , KO KO + A - hWWOX . square box shows magnified from CA3 regions of hippocampus from genotypes . F , G . Graphs represents of GFAP ( F ) Iba1 ( G ) positive from shown in E . number of GFAP or Iba1 were counted from 3 identical sagittal sections from CA3 region per group ( n = 3 per each genotype ) .

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Immunofluorescence staining with anti-GFAP (shown in green) and anti-Iba1 (shown in magenta) antibodies showing the expression of astrocytes and microglia at P19 from WT, KO and KO+A-hWWOX. The square white box shows the magnified area from CA3 regions of hippocampus from all the genotypes. F, G. Graphs represents the quantification of GFAP (F) and Iba1 (G) positive cells from the images shown in E. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections from CA3 region per group (n=3 per each genotype).


H . Immunocytochemical showing astrocytes ( anti - GFAP ) microglia ( anti - Iba1 ) in CA3 region of hippocampus from WT ( n = 3 ) rescued ( KO + AAV - hWWOX ) ( n = 3 ) mice at 9 months . Magnified is shown with square box . I , J . Graphs represents of GFAP ( I ) Iba1 ( J ) positive from shown in H . number of GFAP or Iba1 were counted from 3 identical sagittal sections ( paraffin embedded ) from CA3 region per group ( n = 3 per each genotype ) .

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

H. Immunocytochemical images showing astrocytes (anti-GFAP) and microglia (anti-Iba1) in CA3 region of hippocampus from WT (n=3) and rescued (KO+AAV-hWWOX) (n=3) mice at 9 months. Magnified area is shown with square box. I, J. Graphs represents the quantification of GFAP (I) and Iba1 (J) positive cells from the images shown in H. The number of GFAP or Iba1 cells were counted from 3 identical sagittal sections (paraffin embedded) from CA3 region per group (n=3 per each genotype).


of whole brain sagittal sections that were immunolabelled with anti - MBP at P17 from indicated mice ( n = 3 for each group ) . MBP ( shown in gray ) in cortex ( green dotted represents border of cortex ) , hippocampus cerebellum is presented ( magnified from top panel ) .

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Images of whole brain sagittal sections that were immunolabelled with anti-MBP at P17 from indicated mice (n=3 for each group). MBP staining (shown in gray) in cortex (green dotted line represents the border of the cortex), hippocampus and cerebellum is presented (magnified from the top panel).


Sagittal section of brain ( P17 ) tissues immunostained for CC1 ( green ) PDGFRα ( magenta ) . showing increased number of matured oligodendrocytes in corpus callosum of Wwox null after treatment with AAV - mWwox compared to Wwox null injected with AAV - GFP . Graph represents of CC1 PDGFRα positive in corpus callosum ( 0 . 5 mm2 ) of WT ( n = 3 ) , KO ( n = 3 ) KO + A - mWwox ( n = 3 ) counted from three similar brain sagittal sections per mouse in each genotype .

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

Sagittal section of the brain (P17) tissues immunostained for CC1 (green) and PDGFRα (magenta). Images showing increased number of matured oligodendrocytes in corpus callosum of the Wwox null after treatment with AAV-mWwox compared to Wwox null injected with AAV-GFP virus.   Graph represents the quantification of CC1 and PDGFRα positive cells in corpus callosum (area 0.5 mm2) of WT (n=3), KO (n=3) and KO+A-mWwox (n=3) counted from three similar brain sagittal sections per mouse in each genotype.


Electron micrograph ( EM ) from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9 - hSynI - mWwox - injected mice compared to Wwox - null mice ( KO ) at P17 . Graph showing average number of myelinated axons in corpus callosum from WT ( n = 3 ) , KO ( n = 3 ) or KO mice injected with AAV9 - hSynI - mWwox ( n = 3 ) per field of view ( FOV ) . g - showing reduced myelin thickness of axons in corpus callosum KO ( n = 3 ) compared to KO + AAV - Wwox ( n = 3 ) . Axon diameter myelin thickness was calculated from electron micrograph of corpus callosum ( total no of axons n = 350 per each group ) .

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

Electron micrograph (EM) images from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9-hSynI-mWwox-injected mice compared to Wwox-null mice (KO) at P17.   Graph showing average number of myelinated axons in corpus callosum from WT (n=3), KO (n=3) or KO mice injected with AAV9-hSynI-mWwox (n=3) per field of view (FOV).   g-ratio analysis showing reduced myelin thickness of axons in corpus callosum KO (n=3) compared to the KO+AAV-Wwox (n=3). Axon diameter and myelin thickness was calculated from the electron micrograph images of corpus callosum (total no of axons n=350 per each group).


EM showing increased number of myelinated axons in optic nerve in AAV9 - hSynI - mWwox injected mice compared to KO mice at P17 . Graph showing average number of myelinated axons ( counted per FOV ) in optic nerve from WT ( n = 3 ) , KO ( n = 3 ) KO + AAV - mWwox ( n = 3 ) . g - ' s of myelinated axons from optic nerves showing improved myelin thickness ( less g - ) after WWOX restoration ( KO + AAV - mWwox ) compared to KO alone ( n = 3 per genotype , total no of axons n = 200 per each group ) .

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

EM images showing increased number of myelinated axons in optic nerve in AAV9-hSynI-mWwox injected mice compared to KO mice at P17.   Graph showing the average number of myelinated axons (counted per FOV) in optic nerve from WT (n=3), KO (n=3) and KO+AAV-mWwox(n=3).   g-ratio's of myelinated axons from optic nerves showing improved myelin thickness (less g-ratio) after WWOX restoration (KO+AAV-mWwox) compared to KO alone (n=3 per genotype, total no of axons n=200 per each group).


Representative of open field showing tracking pattern ( open arena ) of WT ( , n = 7 , n = 6 ) AAV - mWwox - injected KO mice ( n = 7 , n = 5 ) at 8 - 10 . Dot plot graphs represent velocity ( B ) , total travelled ( C )

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Representative images of open field test showing tracking pattern (open arena) of WT (females, n=7, males n=6) and AAV-mWwox-injected KO mice (females n=7, males n=5) at 8-10 weeks. Dot plot graphs represent the velocity (B), total distance travelled (C)


A . GAPDH smFISH IF using anti - G3BP antibody ( G3BP ) anti - Dcp1b antibody ( DCP1 ) in A549 ( RL - WT ) or A549 RL - KO either mock transfected ( mock ) or transfected with poly ( I : C ) ( PIC ) for 5 hours . Scale bar 10 microns . B . Graph of fraction of G3BP foci with different volumes in PIC treated RL - WT RL - KO . C . Number of DCP1 foci per in PIC treated RL - WT RL - KO relative to number in mock treated . D . Average volume of Dcp1 foci in mock PIC treated RL - WT RL - KO .

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

A. GAPDH smFISH and IF analysis using anti-G3BP antibody (G3BP) and anti-Dcp1b antibody (DCP1) in A549 cells (RL-WT) or A549 RL-KO cells either mock transfected (mock) or transfected with poly(I:C) (PIC) for 5 hours. Scale bar 10 microns. B. Graph of the fraction of G3BP foci with different volumes in PIC treated RL-WT and RL-KO cells. C. Number of DCP1 foci per cell in PIC treated RL-WT and RL-KO cells relative to the number in mock treated cells. D. Average volume of individual Dcp1 foci in mock and PIC treated RL-WT and RL-KO cells.


A . Western of ( N ) or ( C ) fractions from whole lysates ( W ) from A549 ( RL WT ) , RNase L knock out A549 ( RL KO ) , or A549 with either wild - type RNase L ( NLS - RL - WT ) or RNase L - R667A ( NLS - RL - CM ) fused to a localization signal . Short long exposure with anti - RL antibody . GAPDH used as marker . Histone H3 used as marker . B . Graph depicting fraction of RNase L found in nucleus or cytoplasm . Mean of two experiments with experiment values plotted .

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

A. Western analysis of nuclear (N) or cytoplasmic (C) fractions from whole cell lysates (W) from A549 cells (RL WT), RNase L knock out A549 cells (RL KO), or A549 cells with either wild-type RNase L (NLS-RL-WT) or RNase L-R667A (NLS-RL-CM) fused to a nuclear localization signal sequence. Short and long exposure with anti-RL antibody. GAPDH protein used as cytoplasmic marker. Histone H3 protein used as nuclear marker. B. Graph depicting the fraction of RNase L found in the nucleus or cytoplasm. Mean and SEM of two experiments with individual experiment values plotted.


C . FISH with probes to GAPDH mRNA or oligo ( dT ) to detect poly ( A ) + in A549 without or with either NLS - RL - WT or NLS - RL - CM either mock transfected or transfected with poly ( I : C ) for 4 hours . Scale bar 5 micron .

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

C. FISH with probes to GAPDH mRNA or oligo(dT) to detect poly(A)+ RNA in A549 cells without or with either NLS-RL-WT or NLS-RL-CM either mock transfected or transfected with poly(I:C) for 4 hours. Scale bar 5 micron.


D . Graph depicting median value of of oligo ( dT ) in nuclei divided by volume in indicated types either mock transfected or transfected with poly ( I : C ) for 4 hours . At least 43 nuclei analyzed per condition .

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D. Graph depicting median and the value of the intensity of oligo(dT) fluorescence in individual nuclei divided by nuclear volume in the indicated cell types either mock transfected or transfected with poly(I:C) for 4 hours. At least 43 nuclei analyzed per condition.


of granules in A549 with RNase L targeted to nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . A . FISH to detect poly ( A ) + nucleolar - localized snoRD3A . IF to detect nucleolar granular component nucleophosmin ( NPM1 ) . B . Graph of mean value of average volume of snoRD3A foci in nuclei . 17 nuclei mock treated , 31 nuclei PIC treated . C . Fraction of nuclei with NPM1 enriched in ring structures classified as granular component assemblies or dispersed in nucleoplasm . 17 nuclei mock treated , 31 nuclei PIC treated .

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

Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. A. FISH analysis to detect poly(A)+ RNA and nucleolar-localized snoRD3A RNA. IF analysis to detect nucleolar granular component protein nucleophosmin (NPM1). B. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 17 nuclei mock treated cells, 31 nuclei PIC treated cells. C. Fraction of nuclei with NPM1 protein enriched in ring structures classified as granular component assemblies or dispersed in nucleoplasm. 17 nuclei mock treated cells, 31 nuclei PIC treated cells.


of granules in A549 with RNase L targeted to nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . D . IF to detect dense fibrillar component , ribosome processing factor 1 ( RPF1 ) . E . Graph of mean value of average volume of snoRD3A foci in nuclei . 43 nuclei mock treated , 69 nuclei PIC treated . F . Graph of mean value of average volume of RPF1 foci in 43 mock treated 69 nuclei PIC treated .

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

Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. D. IF analysis to detect dense fibrillar component protein, ribosome processing factor 1 (RPF1). E. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 43 nuclei mock treated cells, 69 nuclei PIC treated cells. F. Graph of the mean and value of the average volume of RPF1 foci in 43 mock treated cells and 69 nuclei PIC treated cells.


of granules in A549 with RNase L targeted to nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . G . FISH to detect poly ( A ) + speckle - localized MALAT1 IF with anti - sc35 antibody to detect speckle SRRM2 . H . Graph of mean value of of MALAT1 signal divided by volume of nuclei . 51 nuclei mock treated , 56 nuclei PIC treated . I . Graph of median value of volume of SRRM2 foci in nuclei . Mock 1526 SRRM2 foci in 51 nuclei . PIC 136 SRRM2 foci in 20 nuclei that contain < 10 SRRM2 foci .

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

Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. G. FISH analysis to detect poly(A)+ RNA and nuclear speckle-localized MALAT1 RNA and IF analysis with anti-sc35 antibody to detect nuclear speckle protein SRRM2. H. Graph of the mean and value of the intensity of MALAT1 signal divided by the nuclear volume of individual nuclei. 51 nuclei mock treated cells, 56 nuclei PIC treated cells. I. Graph of the median and value of the volume of individual SRRM2 foci in nuclei. Mock 1526 SRRM2 foci in 51 nuclei. PIC 136 SRRM2 foci in 20 nuclei that contain <10 SRRM2 foci.


of granules in A549 with RNase L targeted to nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . J . FISH to detect poly ( A ) + IF to detect Cajal coilin . K . Graph depicting fraction of nuclei in mock PIC treated with decreased oligo ( dT ) with different distributions of coilin . 90 nuclei in mock treated , 192 nuclei in PIC treated .

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

Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. J. FISH analysis to detect poly(A)+ RNA and IF analysis to detect Cajal body protein coilin. K. Graph depicting the fraction of nuclei in mock and PIC treated cells with decreased oligo(dT) with different distributions of coilin protein. 90 nuclei in mock treated cells, 192 nuclei in PIC treated cells.


of A549 with NLS - RL - WT or NLS - RL - CM either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . A . FISH to detect poly ( A ) + IF against FUS . are a single 0 . 2 micron Z section .

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

Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. A. FISH analysis to detect poly(A)+ RNA and IF analysis against FUS protein. Images are a single 0.2 micron Z section.


of A549 with NLS - RL - WT or NLS - RL - CM either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . B . Graph depicting fraction of nuclei with FUS dispersed in nucleoplasm or in foci in with reduced oligo ( dT ) signal . nuclei ( total ) in which was degraded ( N = 64 ) or same nuclei classified as to whether or not they contained residual poly ( A ) foci . Nuclei with poly ( A ) foci ( N = 14 ) . Nuclei without poly ( A ) foci ( N = 50 ) .

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

Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. B. Graph depicting the fraction of nuclei with FUS dispersed in the nucleoplasm or in foci in cells with reduced nuclear oligo(dT) signal. All the nuclei (total) in which nuclear RNA was degraded (N=64) or the same nuclei classified as to whether or not they contained residual poly(A) foci. Nuclei with poly(A) foci (N=14). Nuclei without poly(A) foci (N=50).


of A549 with NLS - RL - WT either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . C . FISH to detect poly ( A ) + IF against mediator , MED1 , to detect super - enhancer condensates . D . Number of MED1 foci in mock PIC treated NLS - RL - WT . E . Average volume of MED1 foci in mock PIC treated NLS - RL - WT .

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

Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. C. FISH analysis to detect poly(A)+ RNA and IF analysis against mediator complex protein, MED1, to detect super-enhancer condensates. D. Number of MED1 foci in mock and PIC treated NLS-RL-WT cells. E. Average volume of individual MED1 foci in mock and PIC treated NLS-RL-WT cells.


of A549 with NLS - RL - WT either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . F . FISH to detect poly ( A ) + IF against BRD4 , to detect super - enhancer condensates . G . Number of BRD4 foci in mock PIC treated NLS - RL - WT . H . Average volume of BRD4 foci in mock PIC treated NLS - RL - WT .

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

Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. F. FISH analysis to detect poly(A)+ RNA and IF analysis against BRD4, to detect super-enhancer condensates. G. Number of BRD4 foci in mock and PIC treated NLS-RL-WT cells. H. Average volume of individual BRD4 foci in mock and PIC treated NLS-RL-WT cells.


B ) Volcano plot of quantified . Log2 fold change was determined as difference between mean LFQ of four replicates of telomeric to control p - values were calculated with a Welch t - . above threshold p = 0 . 05 , s0 = 1 , c = 0 . 5 ( dashed ) are considered enriched . Abbreviations : LFQ : Label free

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

B) Volcano plot of quantified proteins. Log2 fold change was determined as the difference between the mean LFQ intensity of the four replicates of telomeric to control sequence and p-values were calculated with a Welch t-test. Proteins above the threshold p=0.05, s0=1, c=0.5 (dashed line) are considered enriched. Abbreviations: LFQ: Label free quantification


D ) of LFQ of enriched at baits . Telomere interactors are indicated on right side of . Color code indicates measured log2 LFQ intensities in telomere pull - down . binding are marked with a black box on left side of figure . Abbreviations : LFQ : Label free RBP : binding

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

D) Heatmap of LFQ intensity of proteins enriched at DNA baits. Telomere interactors are indicated on the right side of the heatmap. Color code indicates measured log2 LFQ intensities in the telomere pull-down. RNA binding proteins are marked with a black box on the left side of the figure. Abbreviations: LFQ: Label free quantification RBP: RNA binding protein


E ) Critically short telomeres arise after 90 population doublings in absence of telomerase ( tlc1 ) . Genomic was extracted at indicated population doublings digested with XhoI . A radioactive probe was used to recognize telomeric in a southern blot . Abbreviations : PD : population doubling .

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

E) Critically short telomeres arise after 90 population doublings in the absence of telomerase (tlc1). Genomic DNA was extracted at indicated population doublings and digested with XhoI. A radioactive probe was used to recognize telomeric DNA in a southern blot. Abbreviations: PD: population doubling.


F ) tlc1 reach senescence at PD 80 - 100 . % viability of indicated strains after propagation in YPD is indicated at different population doublings n = 3 . Asterisk indicates population doubling used in G ) to determine binding of Npl3 to short telomeres by TAP - ChIP . 3 independent spores from indicated genotypes were used . Population doublings are counted from germination of meiotic products hence begin at approx . PD40 . Abbreviations : TAP : tandem affinity purification

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

F) tlc1 cells reach senescence at PD 80-100. %viability of indicated strains after propagation in YPD is indicated at different population doublings n=3. Asterisk indicates population doubling used in G) to determine binding of Npl3 to short telomeres by TAP-ChIP. 3 independent spores from the indicated genotypes were used. Population doublings are counted from germination of meiotic products and hence begin at approx. PD40. Abbreviations: TAP: tandem affinity purification tag


G ) Npl3 - TAP associates strongly to short telomeres . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - n = 3 ( paired t - one * p < 0 . 05 , * * p < 0 . 01 ) . PD90 refers to 90 population doublings in absence of TLC1 ( telomerase subunit ) . no tag control serves as an indicator of non - specific background signal . Abbreviations : TAP : tandem affinity purification tag , PD : population doubling .

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

G) Npl3-TAP associates strongly to short telomeres. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). PD90 refers to 90 population doublings in the absence of TLC1 (telomerase RNA subunit). The no tag control serves as an indicator of non-specific background signal. Abbreviations: TAP: tandem affinity purification tag, PD: population doubling.


A ) Telomere interactors were identified plotted as a volcano plot as in Figure 1 A , B using tlc1 extracts . Interactors were identified using untreated extracts from tlc1 ( left panel ) or digested with RNase A RNase H ( right panel ) .

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

A) Telomere interactors were identified and plotted as a volcano plot as in Figure 1 A, B and using tlc1 protein extracts. Interactors were identified using untreated protein extracts from tlc1 cells (left panel) or digested with RNase A and RNase H (right panel).


B ) Npl3 associates to TERRA . Cross - linked samples from indicated strains were subjected to Npl3 - TAP RIP . Enrichment of indicated RNAs was determined by quantitative PCR on pulled - down reverse - transcribed . Data represents mean % input + / - n = 3 ( unpaired t - two * p < 0 . 05 , * * p < 0 . 01 ) .

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

B) Npl3 associates to TERRA. Cross-linked samples from the indicated strains were subjected to Npl3-TAP RIP. Enrichment of the indicated RNAs was determined by quantitative PCR on pulled-down reverse-transcribed RNA. Data represents mean % input +/- SEM n=3 (unpaired t-test two tailed *p<0.05, **p<0.01).


Npl3 associates strongly to telomeres in absence of Rat1 . C ) Rat1 - AID tagged is strongly degraded following 1h of 1mM IAA treatment . are determined using anti - FLAG ( Rat1 - AID ) anti - actin antibodies . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron .

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

Npl3 associates strongly to telomeres in the absence of Rat1. C) The Rat1-AID tagged protein is strongly degraded following 1h of 1mM IAA treatment. Protein levels are determined using anti-FLAG (Rat1-AID) and anti-actin antibodies. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.


Npl3 associates strongly to telomeres in absence of Rat1 . D ) TERRA increase in expressing Rat1 - AID variant after 1h treatment with 1mM IAA . TERRA were determined by quantitative PCR on reverse - transcribed RNAs after phenol - chloroform extraction . are normalized to 7S ncRNA . Data represents fold increase to corresponding - IAA samples n = 2 . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron .

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

Npl3 associates strongly to telomeres in the absence of Rat1. D) TERRA levels increase in cells expressing the Rat1-AID variant after 1h treatment with 1mM IAA. TERRA levels were determined by quantitative PCR on reverse-transcribed RNAs after phenol-chloroform extraction. RNA levels are normalized to 7S ncRNA levels. Data represents fold increase to corresponding -IAA samples n=2. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.


Npl3 associates strongly to telomeres in absence of Rat1 . E ) Npl3 - TAP associates strongly to telomeres after 1mM IAA treatment in with Rat1 - AID . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - n = 3 ( paired t - one * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron .

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

Npl3 associates strongly to telomeres in the absence of Rat1. E) Npl3-TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1-AID. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.


F - G ) Npl3 associates to telomeres in an R - loop dependent manner . E ) RNH1 was overexpressed from a galactose - inducible promoter . are determined using anti - HA anti - actin antibodies . F ) Npl3 - TAP associates to telomeres in an R - loop - dependent manner . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - n = 3 ( paired t - one * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations : eV : empty vector control , oE :

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

F-G) Npl3 associates to telomeres in an R-loop dependent manner. E) RNH1 was overexpressed from a galactose-inducible promoter. Protein levels are determined using anti-HA and anti-actin antibodies. F) Npl3-TAP associates to telomeres in an R-loop-dependent manner. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: eV: empty vector control, oE: overexpression


A - B ) Npl3 associates to telomeres in early S . A ) cycle profiles of indicated strains . were synchronized in G1 with alpha - factor for 2 . 5h released into cycle at 30 ° C in pre - warmed medium supplemented with indicated HU concentrations . B ) Npl3 - TAP associates strongly to telomeres in early S ( 250 mM HU ) . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - relative to arrested in alpha factor n = 3 ( paired t - two * p < 0 . 05 , * * p < 0 . 01 Abbreviations : ns : not significant HU : hydroxyurea . * p < 0 . 05 , * * p < 0 .

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

A-B) Npl3 associates to telomeres in early S. A) Cell cycle profiles of indicated strains. Cells were synchronized in G1 with alpha-factor for 2.5h and released into the cell cycle at 30°C in pre-warmed medium supplemented with the indicated HU concentrations. B) Npl3-TAP associates strongly to telomeres in early S (250 mM HU). Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to cells arrested in alpha factor n=3 (paired t-test two tailed *p<0.05, **p<0.01 Abbreviations: ns: not significant HU: hydroxyurea. *p<0.05, **p<0.01


C ) NPL3 over expression stabilizes telomeric R - loops . indicated strains were grown on 1 % raffinose 2 % galactose to induce NPL3 over expression . Cross - linked samples were subjected to DRIP . - hybrids were immunoprecipitated using S9 . 6 antibody . R - loop were determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - relative to 250 mM HU eV n = 3 ( paired t - two * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations eV : empty vector control , oE : over expression HU : hydroxyurea . * p < 0 . 05 , * * p < 0 .

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

C) NPL3 over expression stabilizes telomeric R-loops. The indicated strains were grown on 1% raffinose 2% galactose to induce NPL3 over expression. Cross-linked samples were subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to 250 mM HU eV n=3 (paired t-test two tailed *p<0.05, **p<0.01). Abbreviations eV: empty vector control, oE: over expression HU: hydroxyurea. *p<0.05, **p<0.01


D ) Deletion of NPL3 decreases telomeric R - loops . indicated strains were crosslinked subjected to DRIP . - hybrids were immunoprecipitated using S9 . 6 antibody . R - loop were determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - n = 3 . Specificity of S9 . 6 antibody was determined treating a fraction of samples with RNase H .

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

D) Deletion of NPL3 decreases telomeric R-loops. The indicated strains were crosslinked and subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3. Specificity of S9.6 antibody was determined treating a fraction of the samples with RNase H.


E - G ) NPL3 stabilizes R - loops . E ) R - loop dot blot . indicated strains were grown on 1 % raffinose 2 % galactose for 72h subsequently released into 1 % raffinose 2 % galactose or 2 % glucose medium for 6 PDs . R - loop dsDNA were determined using S9 . 6 antibody anti - dsDNA antibodies . Specificity of S9 . 6 antibody was confirmed by treatment with RNase H . Loaded genomic content is indicated at bottom of figure . F ) R - loop dot blot . Data represents R - loop signal as S9 . 6 antibody signal relative to corresponding dsDNA signal n = 3 ( unpaired t - two * p < 0 . 05 ) . G ) Serial dilutions of indicated strains were assayed on 1 % raffinose 2 % galactose media . Plates were imaged after 72h incubation at indicated temperatures . Abbreviations : V : empty vector control , oE : over expression , ds : double - strand ,

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

E-G) NPL3 stabilizes R-loops. E) R-loop dot blot. The indicated strains were grown on 1% raffinose 2% galactose for 72h and subsequently released into 1% raffinose 2% galactose or 2% glucose medium for 6 PDs. R-loop levels and dsDNA levels were determined using the S9.6 antibody and anti-dsDNA antibodies. Specificity of the S9.6 antibody was confirmed by treatment with RNase H. Loaded genomic DNA content is indicated at the bottom of the figure. F) R-loop dot blot quantification. Data represents R-loop signal as the S9.6 antibody signal relative to corresponding dsDNA signal n=3 (unpaired t-test two tailed *p<0.05). G) Serial dilutions of indicated strains were assayed on 1% raffinose 2% galactose media. Plates were imaged after 72h incubation at indicated temperatures. Abbreviations: V: empty vector control, oE: over expression, ds: double-strand,


A ) Serial dilutions of indicated strains were assayed on YPD media . were plated after 30 - 50 generations propagated on YPD agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 72h growth at 30 ° C .

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 B-EXP_ASSAY O O B-EXP_ASSAY O O O O O

A) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C.


B ) Serial dilutions of indicated strains were assayed on YPD media . were plated after 30 - 50 generations propagated on YPD agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 48h growth at 30 ° C .

B) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 48h growth at 30°C.


C ) Serial dilutions of indicated strains were assayed on SC - His media . were plated after 30 - 50 generations propagated on SC - His agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 72h growth at 30 ° C .

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 B-EXP_ASSAY O O B-EXP_ASSAY O O O O O

C) Serial dilutions of indicated strains were assayed on SC-His media. Cells were plated after 30-50 generations propagated on SC-His agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C.


Representative of colon tumors were shown . Scale bars , 10 mm . number , load in each mouse was measured .

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

Representative images of colon tumors were shown. Scale bars, 10 mm. Tumor number, tumor size and tumor load in each mouse was measured.


Histological of colon tumors was shown by hematoxylin eosin ( HE ) . Tumors were microscopically analyzed classified as low or high grade . Histological score was assessed by a pathologist . Scale bars , 25 µm .

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

Histological analysis of colon tumors was shown by hematoxylin and eosin (HE) staining. Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Scale bars, 25 µm.


tissues were stained for Ki67 p - STAT3 . of Ki67 - positive p - STAT3 - positive was quantified . Scale bars , 25 µm .

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

Tumor tissues were stained for Ki67 and p-STAT3. The percentage of Ki67-positive and p-STAT3-positive cells was quantified. Scale bars, 25 µm.


Proportion of CD11b + F4 / 80 + RORγt + / CD45 + lin - was analyzed in colonic LP mLNs among - bearing WT Dectin - 3 - / - mice by flow cytometry .

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

Proportion of CD11b+F4/80+ and RORγt+/CD45+lin- cells was analyzed in colonic LP cells and mLNs among tumor-bearing WT and Dectin-3-/- mice by flow cytometry.


Relative expression of Il - 6 , Il - 22 , Cxcl1 Il - 17a in LP from - bearing WT Dectin - 3 - / - mice were detected using qPCR .

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

Relative expression of Il-6, Il-22, Cxcl1 and Il-17a in LP cells from tumor-bearing WT and Dectin-3-/- mice were detected using qPCR.


number , , load in colons were measured .

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

Tumor number, tumor size, and tumor load in colons were measured.


Tumors were microscopically analyzed classified as low or high grade . Histological score was assessed by a pathologist . tissues were stained for Ki67 . percentages of Ki67 - positive were quantified . Scale bars , 25 µm .

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

Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Tumor tissues were stained for Ki67. The percentages of Ki67-positive tumor cells were quantified. Scale bars, 25 µm.


Mice were treated as described in Fig 1A Fig 2A . Feces were collected from colons in each mouse . Total fungal burden in feces were quantified using qPCR .

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

Mice were treated as described in Fig 1A and Fig 2A. Feces were collected from colons in each mouse. Total fungal burden in feces were quantified using qPCR.


Mice were treated as described in Fig 1A . Fungal ITS2 rDNA gene was performed in each group . Fungal - taxon - at phylum level in feces of mice . Color from blue to red indicates enrichment of fungus .

B-ORGANISM 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 B-TISSUE O B-ORGANISM O O O O O O O O O B-ORGANISM O

Mice were treated as described in Fig 1A. Fungal ITS2 rDNA gene sequence was performed in each group. Fungal-taxon-based analysis at the phylum level in feces of mice. Color from blue to red indicates enrichment of fungus.


Specific fungal burden of C . albicans in feces were quantified using qPCR .

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Specific fungal burden of C.albicans in the feces were quantified using qPCR.


GF mice were orally gavaged with C . albicans ( twice a week , 1 × 107 ) during administration with AOM - DSS ( n = 8 , each group ) . Mice were euthanized on Day 100 , load in each mouse were measured .

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GF mice were orally gavaged with C.albicans (twice a week, 1×107) during administration with AOM-DSS (n=8, each group). Mice were euthanized on Day 100, tumor load in each mouse were measured.


ELISA results for binding assays of Dectin - 3 with C . albicans . Plates were coated with C . albicans then were added with 100 ml / well recombinant Dectin - 3 at indicated concentrations .

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ELISA results for binding assays of Dectin-3 with C. albicans. Plates were coated with C.albicans and then were added with 100 ml/well recombinant Dectin-3 at indicated concentrations.


GFP - C . albicans ( 5 × 106 ) were added onto 1 × 106 BMDMs , incubated at 37 ° C for 1h . Wells were washed fresh media containing fluconazole ( 300 μg / ml ) was added . At 6 hours 18 hours , C . albicans CFU inside BMDMs were photographed calculated by plating on YPD agar . Scale bars , 25 µm .

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GFP-C. albicans (5×106) were added onto 1×106 BMDMs, and incubated at 37°C for 1h. Wells were washed and fresh media containing fluconazole (300 μg/ml) was added. At 6 hours and 18 hours, C. albicans CFU inside BMDMs were photographed and calculated by plating on YPD agar. Scale bars, 25 µm.


Mice were treated as described in Fig . 2A . LP were isolated from each mouse were culture for 48h . Cytokine chemokine production of LP were detected using multiplex cytokine . Color from blue to red indicates enrichment of gene expression .

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Mice were treated as described in Fig.2A. LP cells were isolated from each mouse and were culture for 48h. Cytokine and chemokine production of LP cells were detected using multiplex cytokine assay. Color from blue to red indicates enrichment of gene expression.


mRNA expressions of IL - 22 , β - defensin , Reg3g , Cxcl1 in LP were detected by qPCR .

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mRNA expressions of IL-22, β-defensin, Reg3g, and Cxcl1 in LP cells were detected by qPCR.


LP mLNs were isolated from each mouse were culture for 48h . Production of IL - 22 in LP mLNs were detected by ELISA . LP were isolated from each mouse were culture for 48h . Expression of Il - 17 in LP were detected by qPCR . Production of IL - 17 in LP were detected by ELISA .

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LP cells and mLNs were isolated from each mouse and were culture for 48h. Production of IL-22 in LP cells and mLNs were detected by ELISA. LP cells were isolated from each mouse and were culture for 48h. Expression of Il-17 in LP cells were detected by qPCR. Production of IL-17 in LP cells were detected by ELISA.


level of p - STAT3 STAT3 in colonic epithelial was detected by using Western Blot ( one mouse per lane ) . relative expression of p - STAT3 was calculated .

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Protein level of p-STAT3 and STAT3 in colonic epithelial cells was detected by using Western Blot (one mouse per lane). The relative expression of p-STAT3 was calculated.


WT Dectin - 3 - / - mice were intraperitoneally treated with anti - IL22 antibody or anti - IgG antibody as control during AOM - DSS administration ( n = 5 , each group ) . Mice were euthanized on Day 100 , number , , load in colons were measured .

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WT and Dectin-3-/- mice were intraperitoneally treated with anti-IL22 antibody or anti-IgG antibody as control during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, tumor number, tumor size, and tumor load in colons were measured.


Mice were treated as described in Fig . 1A . LP were isolated from each mouse . proportions of IL - 22 + in CD3 - RORγt + , CD3 - RORγt - , CD3 + RORγt - CD3 + RORγt + were determined by flow cytometry .

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Mice were treated as described in Fig.1A. LP cells were isolated from each mouse. The proportions of IL-22+ cells in CD3-RORγt+, CD3-RORγt-, CD3+RORγt- and CD3+RORγt+ were determined by flow cytometry.


Mice were treated as described in Fig . 1A . LP were isolated from each mouse . proportions of IL - 22 + in NKp46 - RORγt + , NKp46 + RORγt - NKp46 + RORγt + were determined by flow cytometry .

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Mice were treated as described in Fig.1A. LP cells were isolated from each mouse. The proportions of IL-22+ cells in NKp46-RORγt+, NKp46+RORγt- and NKp46+RORγt+ were determined by flow cytometry.


WT Dectin - 3 - / - mice were intraperitoneally treated with anti - CD90 antibody or anti - IgG antibody during AOM - DSS administration ( n = 5 , each group ) . Mice were euthanized on Day 100 , feces were collected in colon from each mouse . Total fungal burden in feces were quantified using 18S rDNA qPCR .

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WT and Dectin-3-/- mice were intraperitoneally treated with anti-CD90 antibody or anti-IgG antibody during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, feces were collected in colon from each mouse. Total fungal burden in feces were quantified using 18S rDNA qPCR.


Mice were treated as described in Fig . 4E colonic LP were isolated . Numbers of IL - 22 + in LP were determined by flow cytometry .

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Mice were treated as described in Fig.4E and colonic LP cells were isolated. Numbers of IL-22+ cells in LP cells were determined by flow cytometry.


Mice were treated as described in Fig . 4E . number , , load histological score in colons were measured .

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Mice were treated as described in Fig.4E. Tumor number, tumor size, tumor load and histological score in colons were measured.


Mice were treated as described in Fig . 2A Fig . 2I . Colonic LP were isolated . Proportion absolute number of IL - 22 + in LP were detected by flow cytometry .

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Mice were treated as described in Fig.2A and Fig.2I. Colonic LP cells were isolated. Proportion and absolute number of IL-22+ cells in LP cells were detected by flow cytometry.


macrophages were isolated from LP in WT Dectin - 3 - / - - bearing mice . mRNA expression of glycolysis - relative genes detected by using qPCR .

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Primary macrophages were isolated from LP cells in WT and Dectin-3-/- tumor-bearing mice. mRNA expression of glycolysis-relative genes detected by using qPCR.


BMDM were stimulated with C . albicans ( 5 × 106 ) , Curdlan , α - mannan for 24h . Glucose uptake , pyruvate level , lactate production , ATP level were determined using kit .

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BMDM cells were stimulated with C.albicans (5×106), Curdlan, and α-mannan for 24h. Glucose uptake, pyruvate level, lactate production, and ATP level were determined using assay kit.


BMDM were stimulated with C . albicans ( 5 × 106 ) for 24h , ECAR OCR were examined .

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BMDM cells were stimulated with C.albicans (5×106) for 24h, and ECAR and OCR were examined.


BMDMs acquired from WT Dectin - 3 - / - mice were stimulated with C . albicans for 24h . Cytokine chemokine production of BMDMs were detected using multiplex cytokine .

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BMDMs acquired from WT and Dectin-3-/- mice were stimulated with C.albicans for 24h. Cytokine and chemokine production of BMDMs were detected using multiplex cytokine assay.


WT - derived BMDMs were stimulated with C . albicans , curldan , or α - mannan in combination with or without 2 - DG ( 2 . 5mM ) for 24h . mRNA expression of Il - 7 were detected by using qPCR . BMDMs were acquired from WT HIF - 1 - / - mice were stimulated with C . albicans , curldan , or α - mannan for 24h . mRNA expression of Il - 7 were detected by using qPCR .

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WT-derived BMDMs were stimulated with C.albicans, curldan, or α-mannan in combination with or without 2-DG (2.5mM) for 24h. mRNA expression of Il-7 were detected by using qPCR. BMDMs were acquired from WT and HIF-1-/- mice and were stimulated with C.albicans, curldan, or α-mannan for 24h. mRNA expression of Il-7 were detected by using qPCR.


ILC3 were sorted from colonic LP were stimulated with indicated cytokines . Productions of IL - 22 were detected by ELISA . mRNA expressions of Il - 22 were detected by qPCR .

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Primary ILC3 cells were sorted from colonic LP cells and were stimulated with indicated cytokines. Productions of IL-22 were detected by ELISA. mRNA expressions of Il-22 were detected by qPCR.


BMDMs were stimulated with C . albicans for 24h supernatant was isolated added to ILC3 . ( 1 ) Supernatant from BMDMs without stimulation . ( 2 ) Supernatant from BMDMs with stimulation of C . albicans . ( 3 ) Supernatant from BMDMs with stimulation of C . albicans IL - 7 antibody . Productions of IL - 22 by ILC3 were detected by ELISA . mRNA expressions of Il - 22 in ILC3 were detected by qPCR .

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BMDMs were stimulated with C.albicans for 24h and the supernatant was isolated and added to primary ILC3 cells. (1) Supernatant from BMDMs without stimulation. (2) Supernatant from BMDMs with stimulation of C.albicans. (3) Supernatant from BMDMs with stimulation of C.albicans and IL-7 antibody. Productions of IL-22 by ILC3 cells were detected by ELISA. mRNA expressions of Il-22 in ILC3 cells were detected by qPCR.


ILC3 were sorted from LPs were stimulated with ( IL - 7 group ) or without IL - 7 ( None group ) . mRNA expressions of relative genes were detected by qPCR .

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Primary ILC3 cells were sorted from LPs and were stimulated with (IL-7 group) or without IL-7 (None group). mRNA expressions of relative genes were detected by qPCR.


ILC3 were sorted in LPs from STAT3fl / fl STAT3fl / fl VillinCre mice were stimulated with IL - 7 . Productions of IL - 22 were detected by ELISA . mRNA expressions of Il - 22 in ILC3 were detected by qPCR .

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Primary ILC3 cells were sorted in LPs from STAT3fl/fl and STAT3fl/fl VillinCre mice and were stimulated with IL-7. Productions of IL-22 were detected by ELISA. mRNA expressions of Il-22 in ILC3 cells were detected by qPCR.


ILC3 were sorted from LPs were stimulated with IL - 7 in combined with FIZC AhR Inh . without stimulation were used as control ( None ) . Production of IL - 22 was detected by ELISA .

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Primary ILC3 cells were sorted from LPs and were stimulated with IL-7 in combined with FIZC and AhR Inh. Cells without stimulation were used as control (None). Production of IL-22 was detected by ELISA.


HEK293T were transiently transfected with indicated expression constructs ( Ctl or AhR ) were stimulated with ( IL - 7 group ) or without IL - 7 ( none group ) . ChIP of interaction of AhR STAT3 to binding sites in il22 promoter with or without IL - 7 , respectively .

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HEK293T cells were transiently transfected with the indicated expression constructs (Ctl or AhR) and were stimulated with (IL-7 group) or without IL-7 (none group). ChIP analysis of the interaction of AhR and STAT3 to binding sites in the il22 promoter with or without IL-7, respectively.


HEK293T were transiently transfected with indicated expression constructs . Whole extracts were subjected to anti - HA immunoprecipitation , were then immunoblotted with HA antibody anti - STAT3 antibody .

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HEK293T cells were transiently transfected with the indicated expression constructs. Whole cell extracts were subjected to anti-HA immunoprecipitation, and were then immunoblotted with HA antibody and anti-STAT3 antibody.


EL4 were transduced with retroviruses encoding AhR STAT3 . IL - 22 mRNA expression was analyzed by qPCR .

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EL4 cells were transduced with retroviruses encoding AhR and STAT3. IL-22 mRNA expression was analyzed by qPCR.


with colon cancer were divided into four groups on stage . mRNA expressions of Dectin - 3 in tissues were detected by qPCR . box represents 25th to 75th percentile , whisker plots represent minimum maximum percentiles . with colon cancer were divided into two groups on fungal burden in feces . mRNA expressions of Dectin - 3 was compared between these two groups .

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Patients with colon cancer were divided into four groups based on tumor stage. mRNA expressions of Dectin-3 in tumor tissues were detected by qPCR. The box represents the 25th to 75th percentile, and the whisker plots represent the minimum and maximum percentiles. Patients with colon cancer were divided into two groups based on fungal burden in the feces. mRNA expressions of Dectin-3 was compared between these two groups.


Overall survival rate was shown by K - M survival curve .

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Overall survival rate was shown by K-M survival curve.


tissues were stained for IL - 22 p - STAT3 . percentages of IL - 22 - positive p - STAT3 - positive were quantified . mRNA expressions of IL22 STAT3 were detected by qPCR . Scale bars , 25 µm .

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Tumor tissues were stained for IL-22 and p-STAT3. The percentages of IL-22-positive and p-STAT3-positive cells were quantified. mRNA expressions of IL22 and STAT3 were detected by qPCR. Scale bars, 25 µm.


B . Blood stage growth rate of mutated lines . A single was injected i . v . into a mouse growth rate was calculated from parasitemia at day 6 - 9 for each mouse ( black dots ) . Bars represent mean of 4 - 7 mice . Significance for ( B ) determined by One - way of variance with Tukeys Multiple Comparison .

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B. Blood stage growth rate of mutated parasite lines. A single parasite was injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9 for each mouse (black dots). Bars represent the mean of 4-7 mice. Significance for (B) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


C . Infected midgut showing oocysts stained with mercurochrome ; scale bar , 100 μm . graph shows oocyst numbers per infected mosquito . Numbers of mosquitoes that were analyzed are indicated above graph . Data derived from at least two independent cage feeds originating from independently infected mice . Box ‐ ‐ whisker plots depict 25 % quantile , median , 75 % quantile , nearest observations within 1 . 5 times interquartile range ( whiskers ) . Significance for ( C ) determined by Kruskal - Wallis with Bonferroni ' s Multiple Comparison .

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C. Infected midgut showing oocysts stained with mercurochrome; scale bar, 100 μm. The graph shows oocyst numbers per infected mosquito. Numbers of mosquitoes that were analyzed are indicated above the graph. Data derived from at least two independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance for (C) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


D . of salivary to midgut sporozoites of indicated lines . Black dots : experiments with 10 - 20 mosquitoes . Bars : mean . Significance for , ( D ) determined by One - way of variance with Tukeys Multiple Comparison .

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D. Ratio of salivary gland to midgut sporozoites of the indicated parasite lines. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Significance for , (D) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


E . Hemolymph sporozoites were isolated observed on glass slides by live imaging . Arrow indicates direction of a motile sporozoite . graph shows fraction of motile hemolymph sporozoites . Black dots : experiments with 10 - 20 mosquitoes . Bars : mean . Significance for ( E ) determined by One - way of variance with Tukeys Multiple Comparison .

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E. Hemolymph sporozoites were isolated and observed on glass slides by live cell imaging. Arrow indicates direction of a motile sporozoite. The graph shows the fraction of motile hemolymph sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Significance for (E) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


F . qRT - PCR of schizonts oocysts of PbMyoA 3 ' dhfs . data points correspond to mean of three technical replicates .

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F. qRT-PCR analysis of schizonts and oocysts of the PbMyoA 3'dhfs parasite line. Individual data points correspond to the mean of three technical replicates.


C . Blood stage growth rate of clonal lines . A single or 100 iRBCs were injected i . v . into a mouse growth rate was calculated from parasitemia at day 6 - 9 . Black dots : mice ; bars : mean . Statistical : ( C ) One - way of variance with Tukeys multiple comparison .

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C. Blood stage growth rate of clonal parasite lines. A single parasite or 100 iRBCs were injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9. Black dots: individual mice; bars: mean. Statistical analysis: (C) One-way analysis of variance with Tukeys multiple comparison test.


D . Proportion of motile ookinetes . Black dots : movie ; bars : mean . Data pooled from three independent biological replicates with 1 - 3 movies taken per experiment . Statistical : ( D ) Kruskal - Wallis with ( D ) Dunn ' s multiple comparisons

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D. Proportion of motile ookinetes. Black dots: Individual movie; bars: mean. Data pooled from three independent biological replicates with 1 - 3 movies taken per experiment. Statistical analysis: (D) Kruskal-Wallis test with (D) Dunn's multiple comparisons test


E . Speed of ookinetes of different lines . Numbers of analysed are shown above graph . Data pooled from three independent experiments ( biological replicates ) . Violin plots show median ( ) quartiles ( dashed lines ) .

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E. Speed of ookinetes of the different lines. Numbers of analysed parasites are shown above the graph. Data pooled from three independent experiments (biological replicates). Violin plots show median (line) and quartiles (dashed lines).


F . Number of oocysts per infected mosquito . Numbers of analyzed mosquitoes are shown above graph . Data pooled from at least three independent cage feeds originating from independently infected mice . Box ‐ ‐ whisker plots depict 25 % quantile , median , 75 % quantile , nearest observations within 1 . 5 times interquartile range ( whiskers ) . Statistical : ( F ) Kruskal - Wallis with ( F ) Bonferroni ' s Multiple Comparison .

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F. Number of oocysts per infected mosquito. Numbers of analyzed mosquitoes are shown above the graph. Data pooled from at least three independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Statistical analysis: (F) Kruskal-Wallis test with (F) Bonferroni's Multiple Comparison test.


A . of hemolymph to midgut sporozoites . Black dots : experiments with 10 - 20 mosquitoes . Bars : mean . B . of salivary to midgut sporozoites . Black dots : experiments with 10 - 20 mosquitoes . Bars : mean . Statistical : ( A ) ( B ) One - way of variance with Tukeys Multiple Comparison .

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A. Ratio of hemolymph to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. B. Ratio of salivary gland to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Statistical analysis: (A) and (B) One-way analysis of variance with Tukeys Multiple Comparison test.


C . Blood stage infection of mice after transmission by mosquito bite . Shown is mean ± standard deviation . D . Blood stage infection of mice after i . v . injection of 1000 sporozoites .

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C. Blood stage infection of mice after transmission by mosquito bite. Shown is the mean ± standard deviation. D. Blood stage infection of mice after i.v. injection of 1000 sporozoites.


AFraction ( left ) speed ( right ) of motile hemolymph sporozoites on glass . Dots correspond to experiments bars indicate mean . Box ‐ ‐ whisker plots depict 25 % quantile , median , 75 % quantile , nearest observations within 1 . 5 times interquartile range ( whiskers ) . Significance for ( A left ) ( B left ) determined by One - way of variance with Tukeys Multiple Comparison . Significance for ( A right ) ( B right ) determined by Kruskal - Wallis with Bonferroni ' s Multiple Comparison .

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AFraction (left) and speed (right) of motile hemolymph sporozoites on glass . Dots correspond to individual experiments and bars indicate the mean. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance for (A left) and (B left) determined by One-way analysis of variance with Tukeys Multiple Comparison test. Significance for (A right) and (B right) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


C . Selected trajectories of 20 manually tracked sporozoites isolated from hemolymph moving over a period of 3 min . Scale bar , 10 μm . D . Selected trajectories of 20 manually tracked sporozoites isolated from salivary moving over a period of 3 min . Scale bar , 10 μm .

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C. Selected trajectories of 20 manually tracked sporozoites isolated from hemolymph and moving over a period of 3 min. Scale bar, 10 μm. D. Selected trajectories of 20 manually tracked sporozoites isolated from salivary glands and moving over a period of 3 min. Scale bar, 10 μm.


A . Scheme depicting setup of optical tweezers for measuring forces that a sporozoite can generate to pull a bead out of an optical trap . bead is placed at apical end of sporozoite . graph shows fraction of sporozoites that pulled bead out of optical trap . Black dots : experiments . Bars : mean . 73 - 131 sporozoites were probed for each . Significance determined by One - way of variance with Tukeys Multiple Comparison .

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A. Scheme depicting the setup of optical tweezers for measuring the forces that a sporozoite can generate to pull a bead out of an optical trap. The bead is placed at the apical end of the sporozoite. The graph shows the fraction of sporozoites that pulled the bead out of the optical trap. Black dots: individual experiments. Bars: mean. 73-131 sporozoites were probed for each line. Significance determined by One-way analysis of variance with Tukeys Multiple Comparison test.