D - Phenotypic of - infiltrating T at day 21 after melanoma inoculation ( Vehicle , n = 5 ; DSF , n = 4 , one had disappeared ) . Data information : , data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . ns , not significant [ multiple t between two groups in ( D )

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D - Phenotypic analysis of tumor-infiltrating T cells at day 21 after melanoma inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Data information: , data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [multiple t tests between the two groups in (D)


immunofluorescence ( E ) of - infiltrating T at day 21 after melanoma inoculation ( Vehicle , n = 5 ; DSF , n = 4 , one had disappeared ) . Scale bars , 20 µm . Statistics data are from 40 × field of view .

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immunofluorescence (E) analysis of tumor-infiltrating T cells at day 21 after melanoma inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Scale bars, 20 µm. Statistics data are from 40× field of view.


F Phenotypic of - infiltrating T at day 21 after melanoma inoculation ( Vehicle , n = 5 ; DSF , n = 4 , one had disappeared ) . Data information : data are representative of 3 independent experiments , mean ± SD .

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F Phenotypic analysis of tumor-infiltrating T cells at day 21 after melanoma inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Data information: data are representative of 3 independent experiments, mean ± SD.


G Phenotypic of IFNγ TNFα production in T isolated from draining lymph nodes at day 19 after melanoma inoculation ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . ns , not significant [ multiple t between two groups in ( G )

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G Phenotypic analysis of IFNγ and TNFα production in T cells isolated from draining lymph nodes at day 19 after melanoma inoculation (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [multiple t tests between the two groups in (G)


H , I growth [ ( H ) , ( n = 5 mice per group ) ] survival curves [ ( I ) , ( n = 5 mice per group ) ] in Vehicle - DSF - treated mice injected s . c . with B16F10 - melanoma followed by intraperitoneal ( i . p . ) injection with PD - 1 antibody on days 0 4 then every third day ( n = 5 ) . Data information : data are representative of 3 independent experiments , mean ± SD . ns , not significant two - way ANOVA in ( H ) , log - rank ( Mantel - Cox ) in ( I ) ] .

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H, I Tumor growth [(H), (n = 5 mice per group)] and survival curves [(I), (n = 5 mice per group)] in Vehicle- and DSF-treated mice injected s.c. with B16F10-melanoma cells followed by intraperitoneal (i.p.) injection with PD-1 antibody on days 0 and 4 and then every third day (n = 5). Data information: data are representative of 3 independent experiments, mean ± SD. ns, not significant two-way ANOVA in (H), log-rank (Mantel-Cox) test in (I)].


A growth in Vehicle - DSF - treated mice injected s . c . with B16F10 melanoma followed by intraperitoneal ( i . p . ) injection with CD8 ( 2A3 ) antibody on days − 1 , 0 , 2 then every second day ( n = 5 ) . Data information : data are representative of 3 independent experiments , mean ± SD . ns , not significant [ two - way ANOVA in ( A )

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A Tumor growth in Vehicle- and DSF-treated mice injected s.c. with B16F10 melanoma cells followed by intraperitoneal (i.p.) injection with CD8(2A3) antibody on days −1, 0, and 2 and then every second day (n = 5). Data information: data are representative of 3 independent experiments, mean ± SD. ns, not significant [two-way ANOVA in (A)


B FACS of cytotoxicity of B16F10 - OVA after coculture with DMSO - or DSF - treated OT - I T ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 . ns , not significant [ two - way

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B FACS analysis of cytotoxicity of B16F10-OVA cells after coculture with DMSO- or DSF-treated primary OT-I T cells (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ****P<0.0001. ns, not significant [two-way ANOVA


C , D growth [ ( C ) , ( n = 5 mice per group ) ] survival curves [ ( D ) , ( n = 10 mice per group ) ] of B16F10 - OVA - bearing mice after adoptive transfer of Vehicle - or DSF - treated OT - I T . Data information : data are representative of 3 independent experiments , mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 . ns , not significant [ two - way ANOVA in ( C ) , log - rank ( Mantel - Cox ) in ( D ) , mean ± SD ] .

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C, D Tumor growth [(C), (n = 5 mice per group)] and survival curves [(D), (n = 10 mice per group)] of B16F10-OVA tumor-bearing mice after adoptive transfer of Vehicle- or DSF-treated primary OT-I T cells. Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ****P<0.0001. ns, not significant [two-way ANOVA in (C), log-rank (Mantel-Cox) test in (D), mean ± SD].


a , Immunoblot of Atg5 LC3 . Brain homogenates were prepared from six - week - old control ( Atg5flox / + ; nestin - Cre ) Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice . Immunoblot was performed using antibodies against Atg5 LC3 . GSK - 3β was used as a loading control . positions of Atg12 - Atg5 conjugate , LC3 - I LC3 - II ( LC3 - PE conjugate ) are indicated . Atg5 monomer was not detected in either lane ( data not shown ) .

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a, Immunoblot analysis of Atg5 and LC3. Brain homogenates were prepared from six-week-old control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice. Immunoblot analysis was performed using antibodies against Atg5 and LC3. GSK-3β was used as a loading control. The positions of the Atg12-Atg5 conjugate, LC3-I and LC3-II (LC3-PE conjugate) are indicated. Atg5 monomer was not detected in either lane (data not shown).


b , A representative control ( Atg5flox / + ; nestin - Cre ) an Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) littermate at three of .

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b, A representative male control (Atg5flox/+; nestin-Cre) and an Atg5flox/flox; nestin-Cre (Atg5flox/flox) littermate at three weeks of age.


c , Left , paw placement records of eight - week - old mice . Right , stride lengths corrected for paw base widths ( stride / width ) of Atg5flox / flox ; nestin - Cre control ( Atg5flox / + ; nestin - Cre ) littermate mice . Values represent means ± s . d . of four mice . Asterisk , P 0 . 01 ( Students t - ) .

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c, Left, paw placement records of eight-week-old mice. Right, stride lengths corrected for paw base widths (stride/width ratio) of Atg5flox/flox; nestin-Cre and control (Atg5flox/+; nestin-Cre) littermate mice. Values represent means ± s.d. of four mice. Asterisk, P 0.01 (Students t-test).


d , Abnormal limb - clasping of an Atg5flox / flox ; nestin - Cre mouse compared with a control mouse ( Atg5flox / + ; nestin - Cre ) when suspended by its tail .

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d, Abnormal limb-clasping of an Atg5flox/flox; nestin-Cre mouse compared with a control mouse (Atg5flox/+; nestin-Cre) when suspended by its tail.


e , Rotarod testing of Atg5flox / + ; nestin - Cre ( open symbols ) Atg5flox / flox ; nestin - Cre ( closed symbols ) mice . One one mouse were analysed for each genotype . time until drop from rod ( rotating at 20 r . p . m . ) is shown .

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e, Rotarod testing of Atg5flox/+; nestin-Cre (open symbols) and Atg5flox/flox; nestin-Cre (closed symbols) mice. One male and one female mouse were analysed for each genotype. The time until drop from the rod (rotating at 20 r.p.m.) is shown.


a , HE - stained sections of cerebral cortex , gracile nucleus cerebellum from control ( Atg5flox / + ; nestin - Cre ) Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice at three months of . Purkinje are indicated with arrowheads . Arrows indicate eosinophilic spheroids , which represent axon swelling . Scale bar , 10 µm .

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a, HE-stained sections of the cerebral cortex, the gracile nucleus and cerebellum from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at three months of age. Purkinje cells are indicated with arrowheads. Arrows indicate eosinophilic spheroids, which represent axon swelling. Scale bar, 10 µm.


b , Immunohistochemistry using an anti - calbindin antibody on cerebellum sections from control ( Atg5flox / + ; nestin - Cre ) Atg5flox / flox ; nestin - Cre mice at six of . Scale bar , 25 µm .

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b, Immunohistochemistry using an anti-calbindin antibody on cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age. Scale bar, 25 µm.


c , Loss of Purkinje in Atg5flox / flox ; nestin - Cre mice . Purkinje were counted in comparable areas for each mouse , three fields were counted in each for each mouse . Data are normalized against values from control mice ( Atg5flox / + ; nestin - Cre ) . Values represent mean ± s . d . of three mice . Asterisk , P 0 . 01 ( t - ) .

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c, Loss of Purkinje cells in Atg5flox/flox; nestin-Cre mice. Purkinje cells were counted in comparable areas for each mouse, and three fields were counted in each area for each mouse. Data are normalized against values from control mice (Atg5flox/+; nestin-Cre). Values represent mean ± s.d. of three mice. Asterisk, P 0.01 (t-test).


d , Apoptotic of granular . Cerebellum sections from control ( Atg5flox / + ; nestin - Cre ) Atg5flox / flox ; nestin - Cre mice at six of were subjected to TUNEL . Nuclei were stained with Hoechst 33258 . Scale bar , 500 µm .

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d, Apoptotic death of granular cells. Cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age were subjected to TUNEL staining. Nuclei were stained with Hoechst 33258. Scale bar, 500 µm.


a , Immunohistochemistry of brain sections from control ( Atg5flox / + ; nestin - Cre ) Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice at six of , stained with an anti - ubiquitin antibody ( 1B3 ) . Scale bar , 10 µm .

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a, Immunohistochemistry of brain sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at six weeks of age, stained with an anti-ubiquitin antibody (1B3). Scale bar, 10 µm.


b , Ubiquitin - positive inclusions in neurons . Sections of medulla from Atg5flox / flox ; nestin - Cre mice at six of were stained with an antibody against NeuN ( a neuron - specific ) ubiquitin . Nuclei were stained with Hoechst 33258 . Scale bar , 10 µm .

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b, Ubiquitin-positive inclusions in neurons. Sections of medulla from Atg5flox/flox; nestin-Cre mice at six weeks of age were stained with an antibody against NeuN (a neuron-specific nuclear protein) and ubiquitin. Nuclei were stained with Hoechst 33258. Scale bar, 10 µm.


c , Immunoelectron microscopy of ubiquitin - positive inclusion bodies . DRG neurons isolated from Atg5 - / - neonates were analysed by immunoelectron microscopy using an anti - ubiquitin antibody . Scale bars , 500 nm .

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c, Immunoelectron microscopy of ubiquitin-positive inclusion bodies. DRG neurons isolated from Atg5-/- neonates were analysed by immunoelectron microscopy using an anti-ubiquitin antibody. Scale bars, 500 nm.


a , Immunohistochemistry of DRG neurons from Atg5 + / + Atg5 - / - mice at different developmental stages ( embryonic day ( E ) 13 . 5 , E15 . 5 or postnatal day ( P ) 0 ) , stained with an antibody directed against ubiquitin . Scale bar , 25 µm .

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a, Immunohistochemistry of DRG neurons from Atg5+/+ and Atg5-/- mice at different developmental stages (embryonic day (E)13.5, E15.5 or postnatal day (P)0), stained with an antibody directed against ubiquitin. Scale bar, 25 µm.


b , Accumulation of Triton - X - 100 - solublepolyubiquitinated in brains of Atg5flox / flox ; nestin - Cre mice . Brain homogenate prepared at indicated times from control ( Atg5flox / + ; nestin - Cre ) Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice were separated into Triton - X - 100 - soluble ( S ) - insoluble ( P ) fractions analysed by immunoblotting using anti - ubiquitin antibodies . Arrowhead indicates stacking gel .

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b, Accumulation of Triton-X-100-solublepolyubiquitinated proteins in the brains of Atg5flox/flox; nestin-Cre mice. Brain homogenate prepared at the indicated times from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice were separated into Triton-X-100-soluble (S) and -insoluble (P) fractions and analysed by immunoblotting using anti-ubiquitin antibodies. Arrowhead indicates the stacking gel.


c , Immunohistochemistry of liver sections from control ( Atg5flox / + ; CAG - Cre ) Atg5flox / flox ; CAG - Cre ( Atg5flox / flox ) mice at four months of , using an anti - ubiquitin antibody . Scale bar , 25 µm .

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c, Immunohistochemistry of liver sections from control (Atg5flox/+; CAG-Cre) and Atg5flox/flox; CAG-Cre (Atg5flox/flox) mice at four months of age, using an anti-ubiquitin antibody. Scale bar, 25 µm.


d , Immunohistochemistry of liver sections from six - week - old Atg5flox / flox ; Mx1 - Cre mice at indicated time points after pIpC injection , using anti - ubiquitin antibodies . Arrowheads indicate ubiquitin - positive inclusion bodies . Scale bar , 25 µm .

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d, Immunohistochemistry of liver sections from six-week-old Atg5flox/flox; Mx1-Cre mice at the indicated time points after pIpC injection, using anti-ubiquitin antibodies. Arrowheads indicate ubiquitin-positive inclusion bodies. Scale bar, 25 µm.


e , Five thousand hepatocytes were randomly selected , number of with diffuse ubiquitin signals , with ( black ) or without ( ) inclusion bodies , were counted .

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e, Five thousand hepatocytes were randomly selected, and the number of cells with diffuse cytosolic ubiquitin signals, with (black) or without (white) inclusion bodies, were counted.


( B ) Immunoblot of WT , cGAS KO , MAVS KO THP1 Data information : Panel ( B ) is representative of two independent biological experiments .

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(B) Immunoblot of WT, cGAS KO, and MAVS KO THP1 cells Data information: Panel (B) is representative of two independent biological experiments.


( C ) THP1 of indicated genotypes were mock infected or infected with VZV for 24 48 hours using transwell described in ( A ) . Infected were quantified by surface for VZV - gE / gI flow cytometry . Data information : Panel ( C ) , show pooled data from four repeats , where each data point represents an independent biological experiment ( n = 4 ± ) . Statistical in panels ( C ) , was paired t - WT : wild type , KO : knockout .

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(C) THP1 cells of the indicated genotypes were mock infected or infected with VZV for 24 and 48 hours using the transwell assay described in (A). Infected cells were quantified by surface staining for VZV-gE/gI and flow cytometry analysis. Data information: Panel (C), show pooled data from four repeats, where each data point represents an independent biological experiment (n=4 ± SEM). Statistical analysis in panels (C), was paired t-tests WT: wild type, KO: knockout.


( D ) RT - qPCR of VZV ORF63 ( immediate early ( IE ) gene ) , ORF54 ( early ( E ) gene ) , ORF40 ( late ( L ) gene ) transcripts in infected as in ( C ) . Graphs show expression relative to GAPDH . Data information : Panel ( D ) show pooled data from three repeats , where each data point represents an independent biological experiment ( n = 3 ± ) . Statistical in panels ( D ) , was paired t - hpi : hours post

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(D) RT-qPCR analysis of VZV ORF63 (immediate early (IE) gene), ORF54 (early (E) gene), and ORF40 (late (L) gene) transcripts in cells infected as in (C). Graphs show expression relative to GAPDH. Data information: Panel (D) show pooled data from three repeats, where each data point represents an independent biological experiment (n=3 ± SEM). Statistical analysis in panels (D), was paired t-tests hpi: hours post infection


( E ) RT - qPCR of IFNB1 , IFI44 , IFIT1 expression in infected as in ( C ) . Graphs show expression relative to GAPDH . Data information : Panel ( E ) show pooled data from three repeats , where each data point represents an independent biological experiment ( n = 3 ± ) . Statistical in panel ( E ) paired t - . hpi : hours post

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(E) RT-qPCR analysis of IFNB1, IFI44, and IFIT1 expression in cells infected as in (C). Graphs show expression relative to GAPDH. Data information: Panel (E) show pooled data from three repeats, where each data point represents an independent biological experiment (n=3 ± SEM). Statistical analysis in panel (E) paired ratio t-tests. hpi: hours post infection


( F ) Activity of Lucia luciferase ( secreted under an IRF3 - dependent promotor ) was determined in supernatants of infected as in ( C ) by QUANTI - Luc . Data information : Panel , ( F ) , show pooled data from four repeats , where each data point represents an independent biological experiment ( n = 4 ± ) . Statistical in panels ( F ) , was paired t - hpi : hours post infection , WT : wild type , KO : knockout .

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(F) Activity of Lucia luciferase (secreted under an IRF3-dependent promotor) was determined in supernatants of cells infected as in (C) by QUANTI-Luc assay. Data information: Panel , (F), show pooled data from four repeats, where each data point represents an independent biological experiment (n=4 ± SEM). Statistical analysis in panels (F), was paired t-tests hpi: hours post infection, WT: wild type, KO: knockout.


( G ) Concentrations of CXCL10 in supernatants of infected as in ( C ) were determined by ELISA . Data information : Panel ( G ) show pooled data from four repeats , where each data point represents an independent biological experiment ( n = 4 ± ) . Statistical in panels ( G ) was paired t - hpi : hours post

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(G) Concentrations of CXCL10 in supernatants of cells infected as in (C) were determined by ELISA. Data information: Panel (G) show pooled data from four repeats, where each data point represents an independent biological experiment (n=4 ± SEM). Statistical analysis in panels (G) was paired t-tests hpi: hours post infection


( A ) HEK293T were transfected with pRL - TK , p125 - F - Luc , expression plasmids for human cGAS human STING , as well as expression plasmids for ORFs . next day luciferase activity was determined . For each ORF a z - value was calculated ORFs sorted by descending values . ( B ) experiment shown in panel ( A ) was repeated three times for each ORF in each experiment a rank was determined according to z - value ( highest z - value = rank 1 ) . Ranks are displayed as a . first row shows results displayed in panel ( A ) . Data information : Panel ( A ) is representative of three independent biological experiments , where each data point represents a technical replicate ( mean ± SD ) . three independent experiments are summarised in panel ( B ) , where data from ( A ) forms first row .

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(A) HEK293T cells were transfected with pRL-TK, p125-F-Luc, expression plasmids for human cGAS and human STING, as well as expression plasmids for individual ORFs. The next day luciferase activity was determined. For each ORF a z-value was calculated and ORFs sorted by descending values. (B) The experiment shown in panel (A) was repeated three times and for each ORF in each experiment a rank was determined according to z-value (highest z-value = rank 1). Ranks are displayed as a heatmap. The first row shows results displayed in panel (A). Data information: Panel (A) is representative of three independent biological experiments, where each data point represents a technical replicate (mean ± SD). The three independent experiments are summarised in panel (B), where the data from (A) forms the first row.


( C ) HEK293T were transfected as in ( A , B ) with expression constructs for VZV ORF9 or empty vector . In parallel , reporter expression was stimulated by co - transfection of RIG - I - CARD plasmid instead of hcGAS hSTING . Data information Panel ( C ) is representing two independent biological experiments , where each data point represents a technical replicate ( mean ± SD ) .

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(C) HEK293T cells were transfected as in (A,B) with expression constructs for VZV ORF9 or empty vector. In parallel, reporter expression was stimulated by co-transfection of the RIG-I-CARD plasmid instead of hcGAS and hSTING. Data information Panel (C) is representing two independent biological experiments, where each data point represents a technical replicate (mean ± SD).


( D ) Western blot of PMA - differentiated THP1 monocytes stably transduced with either V5 - tagged VZV ORF9 or GFP using indicated antibodies . Data information : Panels ( D ) are representing two independent biological experiments . In ( E ) each data point represents a technical replicate .

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(D) Western blot analysis of PMA-differentiated THP1 monocytes stably transduced with either V5-tagged VZV ORF9 or GFP using the indicated antibodies. Data information: Panels (D) are representing two independent biological experiments. In (E) each data point represents a technical replicate.


( E ) THP1 monocytes stably transduced with either VZV ORF9 or GFP were PMA - differentiated transfected with indicated doses of dsDNA . Expression of IFNB1 IFNL1 was assessed by RT - qPCR . Graphs show expression relative to GAPDH . Data information : Panels ( E ) are representing two independent biological experiments . In ( E ) each data point represents a technical replicate .

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(E) THP1 monocytes stably transduced with either VZV ORF9 or GFP were PMA-differentiated and transfected with indicated doses of dsDNA. Expression of IFNB1 and IFNL1 was assessed by RT-qPCR. Graphs show expression relative to GAPDH. Data information: Panels (E) are representing two independent biological experiments. In (E) each data point represents a technical replicate.


( F ) THP1 monocytes stably transduced with either VZV ORF9 or GFP were treated with STING agonist diABZi ( 0 . 005 μM ) . Expression of IFNB1 , IFIT1 IFI44 was assessed by RT - qPCR . Graphs show expression relative to GAPDH . Data information : Panel ( F ) shows pooled data from three two repeats , respectively , where each data point represents an independent biological experiment ( horizontal bars show means ) . Statistical in ( F ) were paired t - .

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(F) THP1 monocytes stably transduced with either VZV ORF9 or GFP were treated with the STING agonist diABZi (0.005 μM). Expression of IFNB1, IFIT1 and IFI44 was assessed by RT-qPCR. Graphs show expression relative to GAPDH. Data information: Panel (F) shows pooled data from three and two repeats, respectively, where each data point represents an independent biological experiment (horizontal bars show means). Statistical analysis in (F) were paired t-tests.


( I ) PMA - differentiated THP1 were co - cultured with VZV - ORF9ecDHFR - infected MeWo for 1h in presence of TMP , washed , incubated for another 24h in presence of TMP . were then washed extensively fresh medium was added that was supplemented or not with TMP . Expression of IFNB1 , IFIT1 , IFI44 were measured by RT - qPCR after a further 24h incubation . Graphs show expression relative to GAPDH . Mean fold changes are indicated . Data information : Panel ( I ) shows pooled data from three repeats , where each data point represents an independent biological experiment . Statistical in ( I ) were paired t - .

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(I) PMA-differentiated THP1 cells were co-cultured with VZV-ORF9ecDHFR-infected MeWo cells for 1h in the presence of TMP, washed, and incubated for another 24h in the presence of TMP. Cells were then washed extensively and fresh medium was added that was supplemented or not with TMP. Expression levels of IFNB1, IFIT1, and IFI44 were measured by RT-qPCR after a further 24h incubation. Graphs show expression relative to GAPDH. Mean fold changes are indicated. Data information: Panel (I) shows pooled data from three repeats, where each data point represents an independent biological experiment. Statistical analysis in (I) were paired t-tests.


( J ) infected treated as in ( I ) with addition of uninfected were analysed by western blotting using indicated antibodies . Data information : Panel ( J ) is representative of three independent experiments .

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(J) Cells infected and treated as in (I) with the addition of uninfected cells were analysed by western blotting using the indicated antibodies. Data information: Panel (J) is representative of three independent experiments.


( A ) HEK293T were transfected with expression plasmids for ORF9 - V5 , human cGAS - FLAG , human STING - HA . next day , were lysed , overexpressed were immunoprecipitated with α - V5 , α - FLAG , α - HA , or IgG isotype control antibody . Input , unbound IP fractions were subjected to immunoblotting using indicated antibodies . Data information : Panels ( A ) are representative of two independent experiments .

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(A) HEK293T cells were transfected with expression plasmids for ORF9-V5, human cGAS-FLAG, and human STING-HA. The next day, cells were lysed, and overexpressed proteins were immunoprecipitated with α-V5, α-FLAG, α-HA, or IgG isotype control antibody. Input, unbound and IP fractions were subjected to immunoblotting using the indicated antibodies. Data information: Panels (A) are representative of two independent experiments.


( B ) THP1 monocytes stably transduced with either VZV FLAG - ORF9 or FLAG - GFP were PMA - differentiated overnight . next day , were lysed ectopically expressed were immunoprecipitated using α - FLAG antibody . Input , unbound IP fractions were subjected to immunoblotting using indicated antibodies . Data information : Panels ( B ) , are representative of three independent experiments .

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(B) THP1 monocytes stably transduced with either VZV FLAG-ORF9 or FLAG-GFP were PMA-differentiated overnight. The next day, cells were lysed and ectopically expressed proteins were immunoprecipitated using α-FLAG antibody. Input, unbound and IP fractions were subjected to immunoblotting using the indicated antibodies. Data information: Panels (B), are representative of three independent experiments.


( C ) HaCaT PMA - differentiated THP1 were infected with WT VZV or VZVORF9 - V5 through co - culture with infected MeWo for 48 hours . were lysed ORF9 was immunoprecipitated using α - V5 antibody . Input IP fractions were subjected to immunoblotting using indicated antibodies . Data information : Panel ( C ) is representative of two ( HaCaT ) three ( THP1 ) independent experiments .

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(C) HaCaT cells and PMA-differentiated THP1 cells were infected with WT VZV or VZVORF9-V5 through co-culture with infected MeWo cells for 48 hours. Cells were lysed and ORF9 was immunoprecipitated using α-V5 antibody. Input and IP fractions were subjected to immunoblotting using the indicated antibodies. Data information: Panel (C) is representative of two (HaCaT) and three (THP1) independent experiments.


( D ) HEK293T were seeded onto glass coverslips were transfected with human cGAS - V5 , FLAG - ORF9 , or both together . next day , were fixed , permeabilised stained using α - V5 - FITC , rabbit - α - FLAG , goat - α - rabbit - AF647 antibodies , DAPI . Mounted coverslips were imaged using confocal microscopy . Scale bars : 15 µm . Data information : Panels ( D ) , are representative of three independent experiments .

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(D) HEK293T cells were seeded onto glass coverslips and were transfected with human cGAS-V5, FLAG-ORF9, or both together. The next day, cells were fixed, permeabilised and stained using α-V5-FITC, rabbit-α-FLAG, and goat-α-rabbit-AF647 antibodies, and DAPI. Mounted coverslips were imaged using confocal microscopy. Scale bars: 15 µm. Data information: Panels (D), are representative of three independent experiments.


( F ) Far western of cGAS - ORF9 interaction . Recombinant FLAG - ORF9 FLAG - GFP were run on an SDS - PAGE gel transferred to a . After renaturation of , was incubated with recombinant human cGAS as a probe , which was detected using α - cGAS antibody . After stripping , on were detected using α - FLAG antibody . Data information : Panels ( F ) are representative of three independent experiments .

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(F) Far western analysis of cGAS-ORF9 interaction. Recombinant FLAG-ORF9 and FLAG-GFP protein were run on an SDS-PAGE gel and transferred to a membrane. After renaturation of proteins, the membrane was incubated with recombinant human cGAS as a probe, which was detected using α-cGAS antibody. After stripping, the proteins on the membrane were detected using α-FLAG antibody. Data information: Panels (F) are representative of three independent experiments.


( G ) Recombinant human cGAS - HA was mixed with recombinant FLAG - ORF9 or FLAG - GFP . were immunoprecipitated using α - HA α - FLAG antibodies . Input , unbound IP fractions were analysed by immunoblotting . Data information : Panels ( G ) are representative of two independent experiments .

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(G) Recombinant human cGAS-HA was mixed with recombinant FLAG-ORF9 or FLAG-GFP. Proteins were immunoprecipitated using α-HA and α-FLAG antibodies. Input, unbound and IP fractions were analysed by immunoblotting. Data information: Panels (G) are representative of two independent experiments.


( B ) HEK293T were transfected with expression plasmids for human cGAS ORF9 truncation mutants . next day were lysed ORF9 were immunoprecipitated using α - FLAG antibody . IP fractions were subjected to immunoblotting using indicated antibodies . Data information : Results shown in panels ( B ) are representative of two independent experiments .

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(B) HEK293T cells were transfected with expression plasmids for human cGAS and ORF9 truncation mutants. The next day cells were lysed and ORF9 proteins were immunoprecipitated using α-FLAG antibody. IP fractions were subjected to immunoblotting using the indicated antibodies. Data information: Results shown in panels (B) are representative of two independent experiments.


( D ) Recombinant cGAS , FLAG - ORF9 , FLAG - GFP , biotinylated VACV70mer dsDNA were incubated in indicated combinations . Recombinant were precipitated using streptavidin beads . Fractions were analysed by immunoblotting . Data information : Results shown are representative of two independent experiments .

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(D) Recombinant cGAS, FLAG-ORF9, FLAG-GFP, and biotinylated VACV70mer dsDNA were incubated in the indicated combinations. Recombinant proteins were precipitated using streptavidin beads. Fractions were analysed by immunoblotting. Data information: Results shown are representative of two independent experiments.


( E ) HEK293T were transfected with expression plasmids for FLAG - GFP or FLAG - ORF9 . next day were lysed , lysate was spiked with biotinylated VACV70mer dsDNA . was precipitated using streptavidin beads fractions were subjected to immunoblotting using indicated antibodies . Data information : Results shown are representative of two independent experiments .

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(E) HEK293T cells were transfected with expression plasmids for FLAG-GFP or FLAG-ORF9. The next day cells were lysed, and the lysate was spiked with biotinylated VACV70mer dsDNA. DNA was precipitated using streptavidin beads and fractions were subjected to immunoblotting using the indicated antibodies. Data information: Results shown are representative of two independent experiments.


( F ) VACV70mer dsDNA was incubated with indicated analysed by agarose gel EMSA . Triangles indicate concentrations of ORF9 ( 0 . 7 , 1 . 4 , 2 . 9 , 5 . 7 , 8 . 6 µM ) , cGAS ( 0 . 6 , 1 . 1 , 2 . 2 , 4 . 5 , 6 . 7 µM ) , ORF9 ( as before ) in presence of 4 . 5 µM cGAS . Data information : Results shown are representative of two independent experiments .

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(F) VACV70mer dsDNA was incubated with indicated proteins and analysed by agarose gel EMSA. Triangles indicate concentrations of ORF9 (0.7, 1.4, 2.9, 5.7, 8.6 µM), cGAS (0.6, 1.1, 2.2, 4.5, 6.7 µM), and ORF9 (as before) in the presence of 4.5 µM cGAS. Data information: Results shown are representative of two independent experiments.


( C ) HEK293T were transfected with expression plasmids for FLAG - GFP or FLAG - ORF9 as indicated . next day were lysed , lysate was spiked with biotinylated VACV70mer dsDNA . was precipitated using streptavidin beads were analysed by immunoblotting using indicated antibodies . Data information : Data shown in panels ( C ) are representative of two independent experiments .

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(C) HEK293T cells were transfected with expression plasmids for FLAG-GFP or FLAG-ORF9 as indicated. The next day cells were lysed, and the lysate was spiked with biotinylated VACV70mer dsDNA. DNA was precipitated using streptavidin beads and proteins were analysed by immunoblotting using the indicated antibodies. Data information: Data shown in panels (C) are representative of two independent experiments.


( D ) HEK293T were transfected with expression plasmids for human cGAS FLAG - GFP or FLAG - ORF9 . next day were lysed , were immunoprecipitated using α - FLAG antibody analysed by immunoblotting using indicated antibodies . Data information : Data shown in panels ( D ) are representative of two independent experiments .

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(D) HEK293T cells were transfected with expression plasmids for human cGAS and FLAG-GFP or FLAG-ORF9. The next day cells were lysed, and proteins were immunoprecipitated using α-FLAG antibody and analysed by immunoblotting using the indicated antibodies. Data information: Data shown in panels (D) are representative of two independent experiments.


( E ) THP1 stably transduced with GFP , ORF9 , or ORF9 - DM were transfected with indicated amounts of E . coli dsDNA . next day , IFNB1 expression was assessed by RT - qPCR . Data information : Panel ( E ) shows pooled data from two repeats , where each data point represents an independent biological experiment ( n = 2 + / - range ) .

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(E) THP1 cells stably transduced with GFP, ORF9, or ORF9-DM were transfected with the indicated amounts of E.coli dsDNA. The next day, IFNB1 expression was assessed by RT-qPCR. Data information: Panel (E) shows pooled data from two repeats, where each data point represents an independent biological experiment (n=2 +/- range).


( F ) In vitro cGAS activity . Recombinant hcGAS was incubated with ATP , GTP , radioactive α32P - ATP at 37 ° C with addition of dsDNA other as indicated ( triangles represent concentrations of 0 . 5 , 1 , 2 , 4 , 6 µM ) . ORF9 - DM was tested only at 0 . 5 , 1 , 2 µM . Reactions were treated with calf - intestinal phosphatase products were analysed by thin - layer chromatography phosphorimaging . ( G ) Signal intensities from ( F ) were determined by densitometry normalized to average BSA signal . Data information : Panel ( F ) is representative of three independent experiments . Panel ( G ) shows pooled data from three repeats , where each data point represents an independent biological experiment ( n = 3 + / - SD ) .

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(F) In vitro cGAS activity assay. Recombinant hcGAS was incubated with ATP, GTP, and radioactive α32P-ATP at 37°C with addition of dsDNA and other proteins as indicated (triangles represent concentrations of 0.5, 1, 2, 4, 6 µM). ORF9-DM was tested only at 0.5, 1, 2 µM. Reactions were treated with calf-intestinal phosphatase and products were analysed by thin-layer chromatography and phosphorimaging. (G) Signal intensities from (F) were determined by densitometry analysis and normalized to the average BSA signal. Data information: Panel (F) is representative of three independent experiments. Panel (G) shows pooled data from three repeats, where each data point represents an independent biological experiment (n=3 +/- SD).


A - F Immunostaining of HL with H2A . B ( brown , indicated by black arrows ) with a counterstain ( blue ) . Human testis ( positive control ) ( A ) , Human tonsils ( negative control ) ( B ) , Nodular sclerosis classical cHL , double with CD15 ( pink ) to identify HRS H2A . B ( brown ) ( C ) , Mixed cellularity cHL ( D ) , Lymphocyte rich cHL ( E ) , Nodular sclerosis HL ( F ) . Scale bar for panels A , B , D - F is 50 µm ; scale bar for panel C is 15 µm .

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A-F Immunostaining of primary HL with H2A.B (brown, indicated by black arrows) with a nuclear counterstain (blue). Human testis (positive control) (A), Human tonsils (negative control) (B), Nodular sclerosis classical cHL, double staining with CD15 (pink) to identify HRS cells and H2A.B (brown) (C), Mixed cellularity cHL (D), Lymphocyte rich cHL (E), Nodular sclerosis HL (F). Scale bar for panels A, B, D-F is 50 µm; scale bar for panel C is 15 µm.


A Relative mRNA expression of H2A . B in L1236 , L428 , HDLM2 HL lines Germinal centre B lymphocytes ( GC ) compared to Ramos B lymphocyte . results are presented as mean ± SD of three biological replicates .

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A Relative mRNA expression levels of H2A.B in L1236, L428, HDLM2 HL cell lines and Germinal centre B lymphocytes (GC) compared to the Ramos B lymphocyte cell line. The results are presented as the mean ± SD of three biological replicates.


D H2A . B interacts with Pol I . Western blot showing co - immunoprecipitation of RPA194 with H2A . B . Pol I subunit RPA194 was immunoprecipitated from L1236 ( left panel ) L428 ( right panel ) extracts . pull - down by RPA194 antibody ( IP ) , pull - down by empty beads or IgG isotype control , 1 % input were loaded for Western blot detection using anti - H2A . B anti - RPA194 antibodies .

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D H2A.B interacts with Pol I. Western blot showing co-immunoprecipitation of RPA194 with H2A.B. The Pol I subunit RPA194 was immunoprecipitated from L1236 (left panel) and L428 (right panel) nuclear extracts. The pull-down by RPA194 antibody (IP), pull-down by empty beads or IgG isotype control, and 1% input protein were loaded for Western blot detection using anti-H2A.B and anti-RPA194 antibodies.


E H2A . B interacts with RPA194 in live . Western blot was performed to detect RPA194 in BioID samples from L1236 ( left panel ) , L428 ( right panel ) . samples were prepared using pull - down with streptavidin beads from lysate of empty vector , BirA * - H2A . B H2A . B - BirA * transduced , which were preincubated with 50 µM biotin for 24 h . positive control was loaded as 1 % input .

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E H2A.B interacts with RPA194 in live cells. Western blot analysis was performed to detect RPA194 in the BioID samples from L1236 cells (left panel), and L428 cells (right panel). The samples were prepared using pull-down with streptavidin beads from the cell lysate of the empty vector, BirA*-H2A.B and H2A.B-BirA* transduced cells, which were preincubated with 50 µM biotin for 24 h. The positive control was loaded as 1% input.


B proliferation in presence ( shNTC ) or during depletion of H2A . B ( shH2A . B ) . results are presented as mean ± SD for five biological replicates . significance of difference was determined by Student ' s t denoted as ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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B Cell proliferation assay in the presence (shNTC) or during depletion of H2A.B (shH2A.B). The results are presented as mean ± SD for five biological replicates. The significance of the difference was determined by Student's t test and denoted as ns (not significant); **, p < 0.01; ***, p<0.001.


E Genome browser screen shots of gene examples ( RPs RPLP1 RPL23A , HDAC5 TAF10 ) that contain H2A . B at their promoters identified by CUT & RUN peak calling whose expression decreased significantly following H2A . B knockdown in - seq . Green purple indicates L1236 L428cells , respectively .

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E Genome browser screen shots of gene examples (RPs RPLP1 and RPL23A, and HDAC5 and TAF10) that contain H2A.B at their promoters identified by the CUT&RUN peak calling and whose expression decreased significantly following H2A.B knockdown in the RNA-seq analysis. Green and purple indicates L1236 and L428cells, respectively.


B Differential exon usage of CD44 gene . C . Differential exon usage of RPL17 gene .

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B Differential exon usage of the CD44 gene. C. Differential exon usage of the RPL17 gene.


D , E Two examples show increased accessibility at promoter peak ( NXF1 ) ( D ) decreased accessibility at peak across intron - exon boundary ( KDM4A ) ( E ) , respectively .

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D, E Two examples show increased accessibility at the promoter peak (NXF1) (D) and decreased accessibility at the peak across the intron-exon boundary (KDM4A) (E), respectively.


Growth inhibitory ( GI50 ) of CY bendamustine in NCI60 panel .

Growth inhibitory concentration (GI50) of CY and bendamustine in the NCI60 cell line panel.


Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer . were taken at 48 h posttreatment .

Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells. Images were taken at 48 h posttreatment.


Cpd A augments damage caused by bendamustine . H1650 were treated with 100 μM Bend , 50 μM Cpd A , 100 μM Bend plus 50 μM Cpd A , or 10 μM CY for 12 h , lysates were blotted for γ - H2AX . Actin was used as a loading control .

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Cpd A augments DNA damage caused by bendamustine. H1650 cells were treated with 100 μM Bend, 50 μM Cpd A, 100 μM Bend plus 50 μM Cpd A, or 10 μM CY for 12 h, and cell lysates were blotted for γ-H2AX. Actin was used as a loading control.


Upper panel , in vitro HDAC1 inhibition using bendamustine CY . Lower panel , summary of CY IC50 against other HDACs .

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Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs.


CY CpdA , but not bendamustine or CpdB , inhibited HDAC activities in cancer . H1650 were treated with 20 μM CY , 20 μM CpdA , 200 μM CpdB , 200 μM Bend , or 10 μM SAHA for 12 h , lysates were blotted for histone acetylation markers . Actin was used as a loading control .

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CY and CpdA, but not bendamustine or CpdB, inhibited HDAC activities in cancer cells. H1650 cells were treated with 20 μM CY, 20 μM CpdA, 200 μM CpdB, 200 μM Bend, or 10 μM SAHA for 12 h, and cell lysates were blotted for histone acetylation markers. Actin was used as a loading control.


Alteration of CY side chain ( Cpd B ) caused loss of HDAC inhibitory activity resulted in reduced ability to induce damage . were treated for 12 h . treated with 200 μM Cpd B 20 μM CY exhibited similar of γ - H2AX . Actin was used as a loading control .

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Alteration of CY side chain (Cpd B) caused loss of HDAC inhibitory activity and resulted in reduced ability to induce DNA damage. Cells were treated for 12 h. Cells treated with 200 μM Cpd B and 20 μM CY exhibited similar levels of γ-H2AX. Actin was used as a loading control.


Summary of lethal dose 50 ( LD50 ) of bendamustine , CY , Cpd A ( HDAC inhibition only ) , Cpd B ( nitrogen mustard only ) against 11 human cancer lines .

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Summary of lethal dose 50 (LD50) of bendamustine, CY, Cpd A (HDAC inhibition only), and Cpd B (nitrogen mustard only) against 11 human cancer cell lines.


Cpd A enhances anticancer activity of bendamustine . H1650 were treated with various doses of bendamustine with or without 40 μM Cpd A ( nontoxic dose , see Fig1C Supplementary Fig S2 ) for 48 h subjected to MTT viability assays .

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Cpd A enhances the anticancer activity of bendamustine. H1650 cells were treated with various doses of bendamustine with or without 40 μM Cpd A (nontoxic dose, see Fig1C and Supplementary Fig S2) for 48 h and subjected to MTT viability assays.


CY , but not bendamustine treatment , led to suppression of CBP , TIP60 , MORF , MSL1 in H1650 . were treated with DMSO , bendamustine ( 350 μM ) , CY ( 15 μM ) , or SAHA ( 10 μM ) for 6 h , mRNA was extracted for qPCR . Data represent mean ± from three independent experiments , statistical significance was determined by unpaired two - t - . * * * P < 0 . 01 .

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CY, but not bendamustine treatment, led to suppression of CBP, TIP60, MORF, and MSL1 in H1650 cells. Cells were treated with DMSO, bendamustine (350 μM), CY (15 μM), or SAHA (10 μM) for 6 h, and mRNA was extracted for qPCR analysis. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. ***P < 0.01.


Drug treatment up to 10 h at concentrations indicated in ( A ) did not cause apoptosis as judged by PARP cleavage . Actin was used as a loading control .

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Drug treatment up to 10 h at the concentrations indicated in (A) did not cause apoptosis as judged by PARP cleavage. Actin was used as a loading control.


ShRNA - mediated suppression of CBP , TIP60 , MORF , MSL1 at mRNA level . Data represent mean ± from three independent experiments , statistical significance was determined by unpaired two - t - . * P < 0 . 1 , * * P < 0 . 05 , * * * P < 0 . 01 .

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ShRNA-mediated suppression of CBP, TIP60, MORF, and MSL1 at mRNA level. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. *P < 0.1, **P < 0.05, ***P < 0.01.


Suppression of CBP , TIP60 , MORF , MSL1 sensitized to bendamustine . Y - axis represents relative resistance calculated from results of GFP competition assays , statistical significance was determined by unpaired two - t - . Data represent mean ± from two independent experiments . * * P < 0 . 05 , * * * P < 0 . 01 .

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Suppression of CBP, TIP60, MORF, and MSL1 sensitized cells to bendamustine. Y-axis represents relative resistance calculated from results of GFP competition assays, and statistical significance was determined by unpaired two-tailed t-test. Data represent mean ± SEM from two independent experiments. **P < 0.05, ***P < 0.01.


A Determination of maximally tolerated dose ( MTD ) of CY at 60 mg / kg . Other drugs were used at MTD according to literature . For each drug , n = 5 mice survived treatment . Data represent mean ± .

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A Determination of maximally tolerated dose (MTD) of CY at 60 mg/kg. Other drugs were used at MTD according to the literature. For each drug, n = 5 and all mice survived treatment. Data represent mean ± SEM.


B CY exhibited enhanced activity against BCR - ABL Arf − / − murine in vitro .

B CY exhibited enhanced activity against BCR-ABL Arf−/− murine ALL cells in vitro.


C , D CY showed superior antitumor activity compared with bendamustine , SAHA , other chemotherapeutic drugs in mice transplanted with BCR - ABL . P - values of CY versus other drugs : NT ( P = 5 . 02E - 06 ) ( C ) , SAHA ( P = 4 . 45E - 06 ) , Bend ( P = 5 . 18E - 06 ) , NT ( P = 1 . 20E - 05 ) ( D ) , VCR ( P = 1 . 13E - 05 ) , Dox ( P = 8 . 08E - 06 ) , AraC ( P = 7 . 62E - 06 ) , CTX ( P = 1 . 13E - 05 ) . Survival statistical was done with Mantel - Cox ( log - rank ) of GraphPad Prism .

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C, D CY showed superior antitumor activity compared with bendamustine, SAHA, and other chemotherapeutic drugs in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 5.02E-06) (C), SAHA (P = 4.45E-06), Bend (P = 5.18E-06), NT (P = 1.20E-05) (D), VCR (P = 1.13E-05), Dox (P = 8.08E-06), AraC (P = 7.62E-06), and CTX (P = 1.13E-05). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.


E Therapeutic effects of CY ( weekly dose ) targeted drugs PP242 ( daily dose ) dasatinib ( daily dose ) in mice transplanted with BCR - ABL . P - values of CY versus other drugs : NT ( P = 3 . 94E - 05 ) , PP242 ( P = 1 . 20E - 05 ) , dasatinib ( P = 0 . 99 ) . Survival statistical was done with Mantel - Cox ( log - rank ) of GraphPad Prism .

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E Therapeutic effects of CY (weekly dose) and targeted drugs PP242 (daily dose) and dasatinib (daily dose) in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 3.94E-05), PP242 (P = 1.20E-05), and dasatinib (P = 0.99). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.


F , G Antitumor activity of CY bendamustine in nude mice models . Mice were inoculated with human NSCLC H460 . When volume reached 200 mm3 , mice were treated with single dose of bendamustine ( 40 mg / kg , MTD ) or CY ( 20 , 40 , 60 mg / kg ) . In ( G ) , tumors were dissected out after 15 days posttreatment .

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F, G Antitumor activity of CY and bendamustine in nude mice models. Mice were inoculated with human NSCLC cell line H460. When tumor volume reached 200 mm3, mice were treated with single dose of bendamustine (40 mg/kg, MTD) or CY (20, 40, 60 mg/kg). In (G), tumors were dissected out after 15 days posttreatment.


Dose curve of CY , fludarabine , bendamustine using fresh CLL samples . Data represent mean ± SD .

Dose curve of CY, fludarabine, and bendamustine using fresh CLL samples. Data represent mean ± SD.


CY preferentially killed CLL over healthy B . Data represent mean ±

CY preferentially killed CLL cells over healthy B cells. Data represent mean ± SD


Toxicity of CY190602 in harboring deletion of chromosome 17p ( including p53 locus ) compared to without del17p . Viability of samples was normalized to background apoptosis for samples . For experiments , viability was determined after 72 h of drug treatment . Data represent mean ± SD .

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Toxicity of CY190602 in patients harboring deletion of chromosome 17p (including p53 locus) compared to patients without del17p. Viability of samples was normalized to background apoptosis for individual patient samples. For all experiments, cell viability was determined after 72 h of drug treatment. Data represent mean ± SD.


B . Rif1 - ∆ C594 retains function to control replication . Growth of a cdc7 - 1 rif1 - ∆ C594 mutant was compared with growth of cdc7 - 1 RIF1 cdc7 - 1 rif1 ∆ strains at 23ºC ( permissive for cdc7 - 1 allele ) , 26ºC ( mild restrictive ) 30ºC ( strict restrictive )

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B. Rif1-∆C594 retains function to control DNA replication. Growth of a cdc7-1 rif1-∆C594 mutant was compared with growth of cdc7-1 RIF1 and cdc7-1 rif1∆ strains at 23ºC (permissive temperature for cdc7-1 allele), 26ºC (mild restrictive temperature) and 30ºC (strict restrictive temperature)


A . Specimen overview of Chromosome VI - left region showing results of ChIP - Seq of Rif1 Rif1 - ∆ C594 . ChIP reads were normalised against reads from corresponding input samples , relative enrichment is plotted for Chromosome VI coordinates 1 - 80 , 000 . Y - axis shows enrichment values ( linear scale , range is 0 to 3 . 5 ) . Values below 1 are shown in grey , values above 1 ( i . e . sequences enriched in ChIP samples ) are coloured blue ( Rif1 ) red ( Rif1 - ∆ C594 ) . Plots show ChIP results from arrested by α - factor ( G1 ) , released from α - factor at 16ºC for 60 min 90 min , or released from α - factor into 0 . 2 M HU for 60 min at 23ºC

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A. Specimen overview of Chromosome VI-left region showing results of ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for Chromosome VI coordinates 1-80,000. Y-axis shows enrichment values (linear scale, range is 0 to 3.5). Values below 1 are shown in grey, and values above 1 (i.e. sequences enriched in ChIP samples) are coloured blue (Rif1) and red (Rif1-∆C594). Plots show ChIP analysis results from cells arrested by α-factor (G1), released from α-factor at 16ºC for 60 min and 90 min, or released from α-factor into 0.2 M HU for 60 min at 23ºC


B . Rap1 - dependent association of Rif1 with promoter regions of Rap1 - controlled genes . ChIP enrichment around PAU3 ( left ) MAM3 ( right ) , both genes whose transcription is controlled by Rap1 . Values above 1 ( i . e . enriched ) shown by overlaid blue red histograms for Rif1 Rif1 - ∆ C594 , respectively . Values below 1 shown in grey

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B. Rap1-dependent association of Rif1 with the promoter regions of Rap1-controlled genes. ChIP enrichment around PAU3 (left) and MAM3 (right), both genes whose transcription is controlled by Rap1. Values above 1 (i.e. enriched) shown by overlaid blue and red histograms for Rif1 and Rif1-∆C594, respectively. Values below 1 shown in grey


A . ChIP - Seq of Rif1 Rif1 - ∆ C594 shows tRNA gene origin binding , with widened peaks at early origins ARS606 ARS607 in HU block . Plots show Chromosome VI genome coordinates 160 , 000 - 210 , 000 . Plot colours here in following Figures are as in Fig 2B . Widened peaks are not observed in unperturbed S phase samples , or for Rif1 - ∆ C594

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A. ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins shows tRNA gene and origin binding, with widened peaks at early origins ARS606 and ARS607 in HU block. Plots show Chromosome VI genome coordinates 160,000- 210,000. Plot colours here and in following Figures are as in Fig 2B. Widened peaks are not observed in unperturbed S phase samples, or for Rif1-∆C594


B . Association of Rif1 - ∆ C594 at replication origins is enhanced in HU block . ChIP enrichment of Rif1 Rif1 - ∆ C594 around early origin ARS1426 ( left ) late origin ARS1412 ( right )

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B. Association of Rif1-∆C594 at replication origins is enhanced in HU block. ChIP enrichment of Rif1 and Rif1-∆C594 around early origin ARS1426 (left) and late origin ARS1412 (right)


C . Differential association of Rif1 Rif1 - ∆ C594 to centromeres . ChIP enrichment of Rif1 Rif1 - ∆ C594 around CEN4 locus

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C. Differential association of Rif1 and Rif1-∆C594 to centromeres. ChIP enrichment of Rif1 and Rif1-∆C594 around the CEN4 locus


A . ChIP - qPCR confirmation of Rif1 Rif1 - ∆ C594 association with replication origins . ChIP was performed using arrested in α - factor ( open bars ) or HU ( grey bars ) , qPCR performed for early origin ARS1426 ( left ) late origin ARS1412 ( right ) . Values shown are ' normalised ChIP efficiencies ' obtained by subtracting value obtained at a control locus ( see Materials Methods ) . Bars indicate averages of two biological replicates , with values from each replicate shown by open circles

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A. ChIP-qPCR confirmation of Rif1 and Rif1-∆C594 association with replication origins. ChIP was performed using cells arrested in α-factor (open bars) or HU (grey bars), and qPCR analysis performed for early origin ARS1426 (left) and late origin ARS1412 (right). Values shown are 'normalised ChIP efficiencies' obtained by subtracting the value obtained at a control locus (see Materials and Methods). Bars indicate the averages of two biological replicates, with values from each replicate shown by open circles


B . Numbers of early late origins associated with Rif1 Rif1 - ∆ C594 peaks . Plot showing numbers of early late origins bound by Rif1 Rif1 - ∆ C594 in G1 HU - blocked , on peak - calling results

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B. Numbers of early and late origins associated with Rif1 and Rif1-∆C594 peaks. Plot showing numbers of early and late origins bound by Rif1 and Rif1-∆C594 in G1 and HU-blocked cells, based on peak-calling results


C . Replication timing programme is intact in rif1 - ∆ C594 . Replication of selected origins at an HU block analysed by BrdU incorporation . were synchronised by α - factor released into medium containing 0 . 2 M HU 1 . 13 mM BrdU . Plots show of total ARS607 , ARS422 . 5 , or ARS1412 pulled down by IP with anti - BrdU , calculated from two biological replicate samples . Bars indicate average of two biological replicates open circles values from each replicate . Insets in ARS422 , 5 ARS1412 panels show same data with Y - axis scales adjusted to 0 - 0 . 03 %

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C. Replication timing programme is intact in rif1-∆C594 cells. Replication of selected origins at an HU block analysed by BrdU incorporation. Cells were synchronised by α-factor and released into the medium containing 0.2 M HU and 1.13 mM BrdU. Plots show the percentage of total ARS607, ARS422.5, or ARS1412 DNA pulled down by IP with anti-BrdU, calculated from two biological replicate samples. Bars indicate the average of two biological replicates and open circles the values from each replicate. Insets in ARS422,5 and ARS1412 panels show the same data with Y-axis scales adjusted to 0 - 0.03%


Expression of YAP / TAZ a smaller ( asterisk ) in different lines , expression was determined by immunoblotting ( IB ) .

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Expression of YAP/TAZ and a smaller protein (asterisk) in different cell lines, protein expression was determined by immunoblotting (IB).


shRNA targeting TAZ , but not YAP , knocked down expression of smaller ( asterisk ) .

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The shRNA targeting TAZ, but not YAP, knocked down the expression of the smaller protein (asterisk).


Antibodies targeting C - terminus YAP / TAZ , such as TAZ ( CST ) YAP / TAZ , effectively pulled down smaller ( asterisk , dubbed as cTAZ hereafter ) in RKO in an immunoprecipitation ( IP ) . cTAZ was not recognized by TAZ ( SA ) , an antibody targeting N - terminus of TAZ .

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Antibodies targeting C-terminus YAP/TAZ, such as TAZ (CST) and YAP/TAZ, effectively pulled down the smaller protein (asterisk, dubbed as cTAZ hereafter) in RKO cells in an immunoprecipitation (IP) assay. cTAZ was not recognized by TAZ (SA), an antibody targeting N-terminus of TAZ.


Exogenous TAZ / YAP was not processed proteolytically to cTAZ . RKO were transfected with indicated plasmids expressing C - terminal HA - tagged TAZ or YAP .

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Exogenous TAZ/YAP was not processed proteolytically to cTAZ. RKO cells were transfected with the indicated plasmids expressing C-terminal HA-tagged TAZ or YAP.


RKO , but not HEK293A , expressed both full - TAZ cTAZ transcripts . RT PCR primers targeting different

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RKO, but not HEK293A cells, expressed both full-length TAZ and cTAZ transcripts. RT PCR primers targeting different regions


Alignment of TAZ cTAZ sequences . cTAZ was derived from Sanger sequencing results of short PCR product ( RKO , F2 : R1 )

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Alignment of TAZ and cTAZ protein sequences. cTAZ protein sequence was derived from the Sanger sequencing results of the short PCR product (RKO, F2:R1)


Knock out of cTAZ in RKO using CRISPR / Cas9 technology . Monoclones ( # 3 # 14 ) were selected .

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Knock out of cTAZ in RKO cells using CRISPR/Cas9 technology. Monoclones (#3 and #14) were selected.


Cluster of expression of WT , cTAZ - / - , cTAZ - / - / GFP - cTAZ put - back RKO . Genes down - up - regulated more than 2 folds ( P < 0 . 05 ) in cTAZ KO were included in cluster . Green red indicate down - up - regulated genes respectively .

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Cluster analysis of RNA expression of WT, cTAZ-/-, and cTAZ-/- /GFP-cTAZ put-back RKO cells. Genes down- and up-regulated more than 2 folds (P < 0.05) in cTAZ KO cells were included in cluster analysis. Green and red indicate down- and up-regulated genes respectively.


cTAZ suppressed expression of ISGs . of selected ISGs in WT , cTAZ - / - , cTAZ - / - / GFP - cTAZ RKO were quantified by qPCR . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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cTAZ suppressed expression of ISGs. RNA levels of selected ISGs in WT, cTAZ-/-, cTAZ-/-/GFP-cTAZ RKO cells were quantified by qPCR. Error bars indicate SD, n =3. **P<0.01; ***P<0.001; Student's t-test.


cTAZ repressed 5 × ISRE - luciferase activity induced by IFN - α . HEK293A were transfected with 5 × ISRE - luciferase reporter along with indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 12 hours . expression of ectopic genes was determined by IB . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; one - way ANOVA was used for statistical .

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cTAZ repressed 5×ISRE-luciferase activity induced by IFN-α. HEK293A cells were transfected with 5×ISRE-luciferase reporter along with the indicated plasmids, treated with or without IFN-α (50 ng/ml) for 12 hours. The expression of ectopic genes was determined by IB. Error bars indicate SD, n =3. ***P<0.001; one-way ANOVA test was used for statistical analysis.


Knockout of cTAZ promoted ISGs expression in presence of IFN - α . WT cTAZ - / - RKO were treated with or without of IFN - α ( 50 ng / ml ) for indicated time , expression of selected genes was quantified by qPCR . WT or cTAZ - / - group was normalized by basal level , respectively . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; Student ' s t - .

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Knockout of cTAZ promoted ISGs expression in the presence of IFN-α. WT and cTAZ-/- RKO cells were treated with or without of IFN-α (50 ng/ml) for indicated time, the expression of selected genes was quantified by qPCR. WT or cTAZ-/- group was normalized by basal level, respectively. Error bars indicate SD, n =3. *P<0.05; **P<0.01; Student's t-test.


STAT1 interacted with cTAZ , interaction was enhanced by IFN - α . HEK293A were transfected with indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 1 hour . IP were performed using Flag antibody .

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STAT1 interacted with cTAZ, and the interaction was enhanced by IFN-α. HEK293A cells were transfected with indicated plasmids, treated with or without IFN-α (50 ng/ml) for 1 hour. IP were performed using Flag antibody.


cTAZ attenuated heterodimerization between STAT1 STAT2 induced by IFN - α . Following transfection , were treated with or without IFN - α ( 50 ng / ml ) for 1 hour subjected to IP assays using Flag antibody .

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cTAZ attenuated heterodimerization between STAT1 and STAT2 induced by IFN-α. Following transfection, cells were treated with or without IFN-α (50 ng/ml) for 1 hour and subjected to IP assays using Flag antibody.


Depletion of cTAZ enhanced interaction between STAT1 STAT2 . RKO lines ( WT # 14 ) were treated with IFN - α as in ( B ) . IP assays were performed using STAT1 antibody .

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Depletion of cTAZ enhanced the interaction between STAT1 and STAT2. RKO cell lines (WT and #14) were treated with IFN-α as in (B). IP assays were performed using STAT1 antibody.


cTAZ attenuated interaction between STAT1 importin α5 ( Im - α5 ) . Following transfection , HEK293A were stimulated using IFN - α IP assays were performed using Flag antibody .

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cTAZ attenuated interaction between STAT1 and importin α5 (Im-α5). Following transfection, HEK293A cells were stimulated using IFN-α IP assays were performed using Flag antibody.


Deletion of cTAZ promoted accumulation of STAT1 upon IFN - α stimulation . RKO ( WT cTAZ - / - ) treated with or without IFN - α ( 50 ng / ml ) for 1 hour , fixed , subjected to IF using STAT1 antibody ( green ) . was labeled by DAPI ( blue ) . Scale bar , 50 μm . was performed using ImageJ software ( right ) . N > C : ; N = C : plus ; N < C : localization .

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Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN-α stimulation. RKO (WT and cTAZ-/-) cells treated with or without IFN-α (50 ng/ml) for 1 hour, fixed, and subjected to IF using STAT1 antibody (green). DNA was labeled by DAPI (blue). Scale bar, 50 μm. Quantification was performed using ImageJ software (right). N>C: nuclear; N=C: nuclear plus cytoplasmic; N<C: cytoplasmic localization.


Deletion of cTAZ promoted accumulation of STAT1 upon IFN - α stimulation . were treated subjected to subcellular fractionation .

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Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN-α stimulation. Cells were treated and subjected to subcellular fractionation.


cTAZ defective in STAT1 - binding failed to block translocation of STAT1 . HEK293A stably overexpressing STAT1 were transfected with indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 1 hour , fixed , subjected to IF Scale bar , 25 μm .

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cTAZ defective in STAT1-binding failed to block nuclear translocation of STAT1. HEK293A stably overexpressing STAT1 were transfected with the indicated plasmids, treated with or without IFN-α (50 ng/ml) for 1 hour, fixed, and subjected to IF Scale bar, 25 μm.


cTAZ repressed antiviral activity of ectopic STAT1 / 2 . HEK293A were transfected with indicated plasmids , infected with gVSV ( MOI = 0 . 01 ) for 12 hours , fixed , subjected to microscopy ( Left ) . Viral infection replication were marked by green . Scale bar , 100 μm . ( Right ) of GFP positive . About 300 ( from 3 different fields ) were analyzed . Error bars indicate SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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cTAZ repressed the antiviral activity of ectopic STAT1/2. HEK293A were transfected with indicated plasmids, infected with gVSV (MOI = 0.01) for 12 hours, fixed, and subjected to microscopy (Left). Viral infection and replication were marked by green fluorescence. Scale bar, 100 μm. (Right) Quantification of GFP positive cells. About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. **P<0.01; ***P<0.001; Student's t-test.


cTAZ and STAT1/2 expression levels of cells


cTAZ effectively repressed antiviral activity of IFN - α . Transfection infection were performed Top : GFP of lysates was measured by a fluorometer . Bottom : viral GFP ectopic cTAZ were detected by IB .

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cTAZ effectively repressed antiviral activity of IFN-α. Transfection and infection were performed Top: GFP intensity of cell lysates was measured by a fluorometer. Bottom: viral GFP and ectopic cTAZ protein levels were detected by IB.


Depletion of cTAZ repressed viral replication . RKO lines ( WT two independent cTAZ - / - clones ) were infected with gVSV ( MOI = 0 . 1 ) for 12 hours . Scale bar , 100 μm . of GFP positive were calculated About 300 ( from 3 different fields ) were analyzed . Error bars indicate SD . * * * P < 0 . 001 ; Student ' s t - .

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Depletion of cTAZ repressed viral replication. RKO cell lines (WT and two independent cTAZ -/- clones) were infected with gVSV (MOI = 0.1) for 12 hours. Scale bar, 100 μm. The ratio of GFP positive cells were calculated About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. ***P<0.001; Student's t-test.


Depletion of cTAZ repressed viral replication . were treated GFP of lysates was measured . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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Depletion of cTAZ repressed viral replication. Cells were treated GFP intensity of cell lysates was measured. Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test.


Depletion of cTAZ repressed viral replication . were treated viral genomic was quantified by qPCR ( normalized to human GAPDH ) . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; Student ' s t - .

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Depletion of cTAZ repressed viral replication. Cells were treated the viral genomic RNA was quantified by qPCR (normalized to human GAPDH). Error bars indicate SD, n = 3. ***P<0.001; Student's t-test.


cTAZ deficiency enhanced ISGs expression . were treated expression of IFIH1 DDX58 were assessed by qPCR . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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cTAZ deficiency enhanced ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; ***P<0.001; Student's t-test.


Overexpression of cTAZ promotes infection . DOX - inducible cTAZ - overexpressing RKO lines were pre - treated with DOX for 48 hours ( 1 μg / ml ) . gVSV infection GFP were performed Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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Overexpression of cTAZ promotes virus infection. DOX-inducible cTAZ-overexpressing RKO cell lines were pre-treated with DOX for 48 hours (1 μg/ml). gVSV infection and GFP Fluorescence intensity quantification were performed Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test.


Overexpression of cTAZ repressed ISGs expression . were treated expression of IFIH1 DDX58 were assessed by qPCR . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; Student ' s t - .

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Overexpression of cTAZ repressed ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; Student's t-test.


gVSV induced expression of cTAZ . RKO were infected with gVSV ( MOI = 0 , 0 . 001 , 0 . 01 , 0 . 01 , 1 ) for 8 hours . expression was determined by IB ( Left ) quantified ( Right ) . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA was used for statistical .

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gVSV induced expression of cTAZ. RKO cells were infected with gVSV (MOI = 0, 0.001, 0.01, 0.01, 1) for 8 hours. Protein expression was determined by IB (Left) and quantified (Right). Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.


IFN - α induced expression of cTAZ . RKO were treated with different doses of IFN - α for 8 hours . expression was determined Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA was used for statistical .

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IFN-α induced expression of cTAZ. RKO cells were treated with different doses of IFN-α for 8 hours. Protein expression was determined Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.


IFN - α induced mRNA level of cTAZ . RKO were treated with different doses of IFN - α for 8 hours . were measured by qPCR . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA was used for statistical .

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IFN-α induced mRNA level of cTAZ. RKO cells were treated with different doses of IFN-α for 8 hours. RNA levels were measured by qPCR. Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.


STAT1 / 2 synergistically activated a cTAZ promoter . HEK293A were transfected with cTAZ reporter with or without STAT . Promoter activity was determined using luciferase , expression was determined by IB . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; Student ' s t - .

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STAT1/2 synergistically activated a cTAZ promoter. HEK293A cells were transfected with cTAZ reporter with or without STAT proteins. Promoter activity was determined using luciferase assay, and protein expression was determined by IB. Error bars indicate SD, n = 3. ***P<0.001; Student's t-test.


STAT1 / 2 binding sites on cTAZ promoter . HEK293A were transfected with cTAZ promoters ( wild type or mutants ) . Luciferase activity responses to STAT1 / 2 expression were measured . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; Student ' s t - .

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STAT1/2 binding sites on cTAZ promoter. HEK293A were transfected with cTAZ promoters (wild type or mutants). Luciferase activity and responses to STAT1/2 expression were measured. Error bars indicate SD, n = 3. **P<0.01; Student's t-test.


STAT1 occupied cTAZ promoter in an IFN - α - sensitive manner . RKO were treated with or without IFN - α ( 50 ng / ml ) for 1 hour subjected to ChIP assays . cTAZ PCR primers were designed to target STAT1 / 2 binding site . ISG15 promoter was included as a positive control .

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STAT1 occupied cTAZ promoter in an IFN-α-sensitive manner. RKO cells were treated with or without IFN-α (50 ng/ml) for 1 hour and subjected to ChIP assays. cTAZ PCR primers were designed to target STAT1/2 binding site. ISG15 promoter was included as a positive control.


of - Seq of differentially expressed lncRNAs ( ≥ 2 - fold change FDR < 0 . 05 ) in mesenchymal ( MES ) 1123 proneural ( PN ) 528 GSCs . GSCs were pre - cultured for 16 h in DMEM / F12 medium with EGF ( 2 ng / ml ) bFGF ( 2 ng / ml ) then followed by co - culturing with or without 20 - μg / ml TGF - β1 for 3 h .

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Heatmap of RNA-Seq analysis of differentially expressed lncRNAs (≥ 2-fold change and FDR< 0.05) in mesenchymal (MES) 1123 and proneural (PN) 528 GSCs. GSCs were pre-cultured for 16 h in DMEM/F12 medium with EGF (2 ng/ml) and bFGF (2 ng/ml) and then followed by co-culturing with or without 20-μg/ml TGF-β1 for 3 h.


Representative of LINC00115 expression in clinical GBM tumors adjacent tissue using RNAscope . are representative of two independent experiments . Scale bars : 50 μm .

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Representative images of LINC00115 expression in clinical GBM tumors and adjacent tissue using RNAscope analysis. Images are representative of two independent experiments. Scale bars: 50 μm.


Kaplan - Meier of with high versus low LINC00115 - expressing GBM tumors from B . Median survival ( in months ) : low , 13 . 9 ; high , 8 . 1 . Black bars , censored data . Data information : , * , p < 0 . 05 , by log - rank .

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Kaplan-Meier analysis of patients with high versus low LINC00115-expressing GBM tumors from B. Median survival (in months): low, 13.9; high, 8.1. Black bars, censored data. Data information: , *, p < 0.05, by log-rank test.


Expression of LINC00115 are higher in LGG ( low grade gliomas ) versus normal brain tissues GBM versus LGG . Expression data of LINC00115 were downloaded from REMBRANDT dataset . Data are presented as mean ± . * * , p < 0 . 01 , * * * , p < 0 . 001 , by two - t .

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Expression levels of LINC00115 are higher in LGG (low grade gliomas) versus normal brain tissues and GBM versus LGG. Expression data of LINC00115 were downloaded from the REMBRANDT dataset. Data are presented as mean ± SEM. **, p < 0.01, ***, p < 0.001, by two-tailed t test.


E to G . Kaplan - Meier of with high versus low LINC00115 - expressing Grade III GBM tumors from REMBRANDT ( E ) or TCGA ( G ) 26 [ ] , GBM from GSE83300 50 [ ] ( F ) datasets . Median survival ( in months ) in E : low , 20 . 2 ; high , 15 . 9 ; in F : low , 19 . 4 ; high , 13 . 0 ; in G : low , 33 . 2 ; high , 25 . 4 . Black bars , censored data . Data information : , * , p < 0 . 05 , by log - rank .

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E to G. Kaplan-Meier analysis of patients with high versus low LINC00115-expressing Grade III and GBM tumors from the REMBRANDT (E) or the TCGA (G) 26[], and GBM from GSE83300 50[] (F) datasets. Median survival (in months) in E: low, 20.2; high, 15.9; in F: low, 19.4; high, 13.0; in G: low, 33.2; high, 25.4. Black bars, censored data. Data information: , *, p < 0.05, by log-rank test.


qRT - PCR of LINC00115 knockdown using two different shRNAs , shL115 - 1 shL115 - 2 in 1123 528 GSCs . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , by One - way

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qRT-PCR analysis of LINC00115 knockdown using two different shRNAs, shL115-1 and shL115-2 in 1123 and 528 GSCs. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, by One-way ANOVA


Effects of LINC00115 knockdown on GSC proliferation ( B ) data are representative of three independent experiments . Error bars , ± SD . * , by two - t - in B .

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Effects of LINC00115 knockdown on GSC proliferation (B) data are representative of three independent experiments. Error bars, ± SD. *, by two-tailed t-test in B.


C . Effects of LINC00115 knockdown on neuro - like sphere formation ( C ) . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , by One - way ANOVA

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C. Effects of LINC00115 knockdown on neuro-like sphere formation (C). Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, by One-way ANOVA in


D . Representative bioluminescence of brains with indicated 528 GSC xenografts expressing shC , shL115 - 1 , or shL115 - 2 at 45 days after implantation . represent results of 5 mice per group of 2 independent experiments . E . of bioluminescence activity in D .

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D. Representative bioluminescence images of brains with indicated 528 GSC tumor xenografts expressing shC, shL115-1, or shL115-2 at 45 days after implantation. Images represent results of 5 mice per group of 2 independent experiments. E. Quantification of the bioluminescence activity in D.


F . Representative hematoxylin eosin ( H & E ) - stained of mouse brain sections with GSC1123 xenografts expressing shC , shL115 - 1 , or shL115 - 2 . represent results of five mice per group of two independent experiments . Scale bars : 1 mm . G . of volume in F .

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F. Representative hematoxylin and eosin (H&E)-stained images of mouse brain sections with GSC1123 xenografts expressing shC, shL115-1, or shL115-2. Images represent results of five mice per group of two independent experiments. Scale bars: 1 mm. G. Quantification of tumor volume in F.


H . Immunofluorescence ( IF ) of Nestin expression in 1123 GSC control LINC00115 knockdown xenograft tumors from F . are representative of two independent experiments . Scale bars : 50 μm .

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H. Immunofluorescence (IF) analysis of Nestin expression in 1123 GSC control and LINC00115 knockdown xenograft tumors from F. Images are representative of two independent experiments. Scale bars: 50 μm.


I . Kaplan - Meier of implantation with 1123 / shC , 1123 / shL115 - 1 or 1123 / shL115 - 2 GSCs . n = 10 . Data information : * * , p < 0 . 01 , by log - rank .

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I. Kaplan-Meier analysis of animal implantation with 1123/shC, 1123/shL115-1 or 1123/shL115-2 GSCs. n = 10. Data information: **, p < 0.01, by log-rank test.


B C . MS2 - RIP followed by qRT - PCR of endogenous microRNAs association with LINC00115 . 12XMS2 empty vector , LINC00115 - 12XMS2 , or LINC00115 - mut - 12XMS2 with Flag - tagged MS2 were co - transfected into 1123 GSCs . Data information data are representative of three independent experiments . Error bars , ± SD . , * * * , p < 0 . 001 , by two - t - .

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B and C. MS2-RIP followed by qRT-PCR analysis of endogenous microRNAs association with LINC00115. 12XMS2 empty vector, LINC00115-12XMS2, or LINC00115-mut- 12XMS2 with Flag-tagged MS2 were co-transfected into 1123 GSCs. Data information data are representative of three independent experiments. Error bars, ± SD. , ***, p < 0.001, by two-tailed t-test.