A subsequence of the 3D - PCR amplicon was used as ( - ) ssDNA oligomer , complementary ( + ) ssDNA and a dU - containing ( ‑ ) ssDNA , where all cytosines were replaced by uracils . These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated .

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

A subsequence of the 3D-PCR amplicon was used as (-) ssDNA oligomer, complementary (+) ssDNA and a dU-containing (‑) ssDNA, where all cytosines were replaced by uracils. These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated.


dHepaRG - TR - A3A cells were infected with HBV at an MOI of 300 virions / cell and at day 7 p . i . transduced with AdV - ISG20 and treated with tetracycline to induce A3A expression as indicated ( ± Tet ( A3A ) ) for 7 days . Cell lysates were analyzed for cccDNA after T5 digest ( B ) and total intracellular HBV DNA ( C ) by qPCR relative to Prnp ( n = 5 - 6 biological replicates from two independent experiments ) .

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

dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell and at day 7 p.i. transduced with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. Cell lysates were analyzed for cccDNA after T5 digest (B) and total intracellular HBV DNA (C) by qPCR relative to Prnp (n=5-6 biological replicates from two independent experiments).


dHepaRG - TR - A3A cells were infected with wildtype HBV ( HBVwt ) or HBV ∆ x at an MOI of 300 virions / cell and transduced on day 7 p . i . with AdV - ISG20 and treated with tetracycline to induce A3A expression as indicated ( ± Tet ( A3A ) ) for 7 days . HBeAg ( E ) was measured by ELISA and cccDNA after T5 digest ( F ) by qPCR relative to Prnp ( n = 6 biological replicates from two independent experiments ) .

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

dHepaRG-TR-A3A cells were infected with wildtype HBV (HBVwt) or HBV∆x at an MOI of 300 virions/cell and transduced on day 7 p.i. with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. HBeAg (E) was measured by ELISA and cccDNA after T5 digest (F) by qPCR relative to Prnp (n=6 biological replicates from two independent experiments).


( E ) Normalized expression levels of the native core promoters . Their activities spanned over a broad range of three orders of magnitude ( promoter constructs contained block 1 . 11 and block 7 . 11 combination as nucleosomal sequences ) . Each color represents a different class of core promoter architecture . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . Individual points represent values over 1 . 5 times the interquartile range . 3 - 4 biological replicate measurements . ( including new cell transfection procedures and measurements of promoter activities ) .

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Normalized expression levels of the native core promoters. Their activities spanned over a broad range of three orders of magnitude (promoter constructs contained block 1.11 and block 7.11 combination as nucleosomal sequences). Each color represents a different class of core promoter architecture. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (including new cell transfection procedures and measurements of promoter activities).


( I ) Scatter plot of expression levels obtained in D . mel . S2 cells by our Luciferase assay pipeline vs mNeonGreen reporter expression in living D . mel . embryos , revealing a high correlation ( Pearson coefficient 0 . 91 ) between the two datasets . Error bars represent standard deviations of 3 - 4 biological replicate measurements .

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

(I) Scatter plot of expression levels obtained in D. mel. S2 cells by our Luciferase assay pipeline vs mNeonGreen reporter expression in living D. mel. embryos, revealing a high correlation (Pearson coefficient 0.91) between the two datasets. Error bars represent standard deviations of 3-4 biological replicate measurements.


( C ) Boxplots depicting block 1 effects for tested core promoters . Effects of different block 7s were merged in each column ( within the same block 1 ) : the median SD is 0 . 66 for developmental promoters compared to 1 . 23 for constitutive promoters ( lower right corner ) ; Wilcoxon rank - sum test * * * p = 3 . 1 × 10 - 4 , significant . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . Individual points represent values over 1 . 5 times the interquartile range . 3 - 4 biological replicate measurements . ( D ) Boxplots depicting block 7 effects for tested core promoters . Effects of different block 1s were merged in each column ( within the same block 7 ) : the median SD is 0 . 54 for developmental promoters compared to 0 . 64 for constitutive promoters ( lower right corner ) ; Wilcoxon rank - sum test p = 0 . 3 , not significant . Block 7 with column " w / o B7 " represents the results obtained from promoters without block 7 sequence . Developmental and constitutive promoters are highlighted in green and red , respectively . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . Individual points represent values over 1 . 5 times the interquartile range . 3 - 4 biological replicate measurements .

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

(C) Boxplots depicting block 1 effects for tested core promoters. Effects of different block 7s were merged in each column (within the same block 1): the median SD is 0.66 for developmental promoters compared to 1.23 for constitutive promoters (lower right corner); Wilcoxon rank-sum test ***p = 3.1×10-4, significant. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (D) Boxplots depicting block 7 effects for tested core promoters. Effects of different block 1s were merged in each column (within the same block 7): the median SD is 0.54 for developmental promoters compared to 0.64 for constitutive promoters (lower right corner); Wilcoxon rank-sum test p = 0.3, not significant. Block 7 with column "w/o B7" represents the results obtained from promoters without block 7 sequence. Developmental and constitutive promoters are highlighted in green and red, respectively. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements.


( B - C ) Comparison of normalized expression levels between wild - type configuration and motif knockouts for two types of core promoters ( developmental : CG8157 ( B ) ; constitutive : RpL5 ( C ) ) . Upper panels : schematic depiction of the wild - type motif compositions ( TTGTT motif in RpL5 is ignored due to its strong overlap with R - INR ) . Two - sample t - test : ns , not significant , * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . 3 - 4 biological replicate measurements .

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

(B-C) Comparison of normalized expression levels between wild-type configuration and motif knockouts for two types of core promoters (developmental: CG8157 (B); constitutive: RpL5 (C)). Upper panels: schematic depiction of the wild-type motif compositions (TTGTT motif in RpL5 is ignored due to its strong overlap with R-INR). Two-sample t-test: ns, not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. 3-4 biological replicate measurements.


( A ) Consensus replacement . Heatmap depicting the mean expression fold changes compared to wild - type expressions after replacing with motif consensus sequences derived by XXmotif . Constitutive and developmental promoters are highlighted in red and green , respectively .

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

(A) Consensus replacement. Heatmap depicting the mean expression fold changes compared to wild-type expressions after replacing with motif consensus sequences derived by XXmotif. Constitutive and developmental promoters are highlighted in red and green, respectively.


( B ) Expression fold changes ( ecdysone inducibility ) versus measurements without ecdysone and grouped by native core promoter sequences . The colors refer to different core promoter architectures . Three types of line are used to indicate the expression fold change with no increase ( y = x ; solid line ) , 2 - fold increase ( y = x + 1 ; dotted line ) and 4 - fold increase ( y = x + 2 ; dashed line ) . Red vertical dashed line : log2 basal expressions = 2 .

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

(B) Expression fold changes (ecdysone inducibility) versus measurements without ecdysone and grouped by native core promoter sequences. The colors refer to different core promoter architectures. Three types of line are used to indicate the expression fold change with no increase (y = x; solid line), 2-fold increase (y = x + 1; dotted line) and 4-fold increase (y = x + 2; dashed line). Red vertical dashed line: log2 basal expressions = 2.


( C ) Heatmap depicting the ecdysone inducibility fold changes caused by individual knockout of motifs in different core promoters . Disrupted INR ( highlighted with the black dotted line rectangle ) had a slightly negative effect on changing the core promoter responsiveness to ecdysone . ( ~ 2 . 3 - fold reduction on average , Wilcoxon rank - sum test p = 2 . 1 × 10 - 5 ) . Constitutive and developmental promoters highlighted in red and green , respectively .

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 O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Heatmap depicting the ecdysone inducibility fold changes caused by individual knockout of motifs in different core promoters. Disrupted INR (highlighted with the black dotted line rectangle) had a slightly negative effect on changing the core promoter responsiveness to ecdysone. (~ 2.3-fold reduction on average, Wilcoxon rank-sum test p = 2.1×10-5). Constitutive and developmental promoters highlighted in red and green, respectively.


( B ) Violin plots displaying RNA - seq readings by FPKM ( normalized Fragments Per Kilobase Million ) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls ( n = 49 , 42 ; unpaired Kolmogorov - Smirnov test ; continuous lines : median ; dashed lines : quartiles ) .

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

(B) Violin plots displaying RNA-seq readings by FPKM (normalized Fragments Per Kilobase Million) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls (n = 49,42; unpaired Kolmogorov-Smirnov test; continuous lines: median; dashed lines: quartiles).


( D ) Mean and SEM of mRNA levels of Xbp1s , Bip / Hspa5 and Chop / Ddit3 were determined in dissected hippocampus of i . p . treated mice by quantitative RT - PCR . ( n = 3 - 4 animals / group . One - way ANOVA followed by Tukey ' s post - test compared tunicamycin treated groups ) .

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

(D) Mean and SEM of mRNA levels of Xbp1s, Bip/Hspa5 and Chop/Ddit3 were determined in dissected hippocampus of i.p. treated mice by quantitative RT-PCR. (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups).


( E ) Animals received bilateral intra - hippocampal injections of 2 μl tunicamcyin ( 5 mg / mL ) or vehicle for 24 h . Graphs indicate mean and s . e . m . of Xbp1s mRNA levels in the hippocampus ( n = 3 - 4 animals / group . One - way ANOVA followed by Tukey ' s post - test compared tunicamycin treated groups .

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

(E) Animals received bilateral intra-hippocampal injections of 2 μl tunicamcyin (5 mg/mL) or vehicle for 24 h. Graphs indicate mean and s.e.m. of Xbp1s mRNA levels in the hippocampus (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups.


( F - G ) Mean and SEM of relative levels of S - nitrosylated IRE1 ( f ) and total IRE1 ( g ) in brains from young and aged mice . Histograms show relative levels of SNO - IRE1 / input IRE1α measured by biotin - switch assay and total IRE1 / GAPDH quantified by densitometry on standard immunoblots for young ( 3 - month - old ) versus aged ( 24 month - old ) mouse brains ( n = 3 , unpaired Student ' s t test ) . Data information :

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

(F-G) Mean and SEM of relative levels of S-nitrosylated IRE1 (f) and total IRE1 (g) in brains from young and aged mice. Histograms show relative levels of SNO-IRE1/input IRE1α measured by biotin-switch assay and total IRE1/GAPDH quantified by densitometry on standard immunoblots for young (3-month-old) versus aged (24 month-old) mouse brains (n = 3, unpaired Student's t test). Data information:


Young ( 3 - 6 months ) , middle aged ( 12 - 14 months ) and aged ( 18 - 20 months ) conditional knockout for IRE1 ( IRE1cKO ) and their littermates flox / flox control animals ( IRE1WT ) were evaluated ( C ) Representative photomicrographs of β - galactosidase staining of hippocampal slices derived from young ( 3 months ) , middle - aged ( 12 months ) and aged ( 22 - 24 months ) of IRE1WT or IRE1cKO animals . Magnification : 20x ; scale bar : 200 μm . Arrows indicate β - galactosidase staining .

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

Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (C) Representative photomicrographs of β-galactosidase staining of hippocampal slices derived from young (3 months), middle-aged (12 months) and aged (22-24 months) of IRE1WT or IRE1cKO animals. Magnification: 20x; scale bar: 200 μm. Arrows indicate β-galactosidase staining.


Young ( 3 - 6 months ) , middle aged ( 12 - 14 months ) and aged ( 18 - 20 months ) conditional knockout for IRE1 ( IRE1cKO ) and their littermates flox / flox control animals ( IRE1WT ) were evaluated ( D ) Mean and SEM of percentage of β - galactosidase staining in CA3 region ( n = 3 - 5 animals / group ) .

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

Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (D) Mean and SEM of percentage of β-galactosidase staining in CA3 region (n = 3-5 animals/group).


( F ) Representative confocal images of dendritic spines ( arrows ) in the CA1 region of middle - aged IRE1WT animals injected with AAV - Mock or AAV - Cre . 60x magnification , ( G ) Mean and SEM of spine density per μm ( n = 3 , 3 ; 4 , 4 animals ) . P computed by unpaired Student ' s t test within each age - matched group .

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

(F) Representative confocal images of dendritic spines (arrows) in the CA1 region of middle-aged IRE1WT animals injected with AAV-Mock or AAV-Cre. 60x magnification, (G) Mean and SEM of spine density per μm (n = 3, 3; 4, 4 animals). P computed by unpaired Student's t test within each age-matched group.


( E ) Representative images of β - galactosidase staining of hippocampal slices derived from aged ( 22 - 24 months ) TgXBP1s and WT animals . ( F ) Mean and SEM of percentage of area stained by β - galactosidase in the hippocampus of aged ( 20 - 24 months ) TgXBP1s compared to littermate controls .

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

(E) Representative images of β-galactosidase staining of hippocampal slices derived from aged (22-24 months) TgXBP1s and WT animals. (F) Mean and SEM of percentage of area stained by β-galactosidase in the hippocampus of aged (20-24 months) TgXBP1s compared to littermate controls.


( D ) Left panel : Representative images showing dendritic spines of aged ( 22 - 24 months ) animals injected with AAV2 - eGFP ( mock ) or with AAV2 - XBP1s ( arrows indicate dendritic spines ) . Young animal dendrite is shown for reference . 60x magnification . Scale bar : 5μm . Right panel : Mean and s . e . m . of spine density per μm for indicated experimental groups ( n = 24 , 53 and 35 dendrites from 3 , 5 and 6 animals ,

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

(D) Left panel: Representative images showing dendritic spines of aged (22-24 months) animals injected with AAV2-eGFP (mock) or with AAV2-XBP1s (arrows indicate dendritic spines). Young animal dendrite is shown for reference. 60x magnification. Scale bar: 5μm. Right panel: Mean and s.e.m. of spine density per μm for indicated experimental groups (n = 24, 53 and 35 dendrites from 3, 5 and 6 animals, respectively


( E ) Left panel : Representative images of β - galactosidase staining of hippocampal slices derived from aged mice injected with AAV2 - Mock or AAV2 - XBP1s ( arrow indicate β - galactosidase staining ) . Magnification 20x ; scale bar , 200 μm . Right panel : Histograms show mean and s . e . m . of percentage of β - galactosidase - stained area . n = 6 , 6 ; unpaired Student ' s t test .

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

(E) Left panel: Representative images of β-galactosidase staining of hippocampal slices derived from aged mice injected with AAV2-Mock or AAV2-XBP1s (arrow indicate β-galactosidase staining). Magnification 20x; scale bar, 200 μm. Right panel: Histograms show mean and s.e.m. of percentage of β-galactosidase-stained area. n = 6, 6; unpaired Student's t test.


( F ) Left panel : Immunofluorescence of γH2aX ( red ) counterstained with nuclei ( blue ) comparing aged mice injected with AAV - Mock or AAV - XBP1s . Magnification : 40x ; Scale bar : 50μm . Right panel : Histograms show mean and s . e . m . of percentage of γH2A - positive cells ; unpaired Student ' s t test comparing the aged groups ( n = 6 , 6 animals / group ; unpaired Student ' s t test ) .

O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-ORGANISM O O B-ORGANISM O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Left panel: Immunofluorescence of γH2aX (red) counterstained with nuclei (blue) comparing aged mice injected with AAV-Mock or AAV-XBP1s. Magnification: 40x; Scale bar: 50μm. Right panel: Histograms show mean and s.e.m. of percentage of γH2A- positive cells; unpaired Student's t test comparing the aged groups ( n = 6, 6 animals/group; unpaired Student's t test).


B FACS analysis of p - S6 in WT mouse splenic CD8 + T cells stimulated for 8 min with α - mouse CD3ε ( 145 - 2C11 ) after pretreatment with DSF ( 5 µM ) for 2 h ( n = 3 ) . Data information : , data are representative of 3 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; ns , not significant [ unpaired t tests for measurements between the two groups in ( B )

O B-EXP_ASSAY O O O O B-GENEPROD O O B-ORGANISM B-TISSUE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FACS analysis of p-S6 in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with DSF (5 µM) for 2 h (n = 3). Data information: , data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (B)


C Representative FACS of p - S6 in WT mouse splenic CD8 + T cells stimulated for 8 min with α - mouse CD3ε ( 145 - 2C11 ) after pretreatment with the indicated concentrations of DSF for 2 h ( n = 2 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; ns , not significant [ unpaired t tests for measurements between the two groups in ( C )

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

C Representative FACS of p-S6 in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with the indicated concentrations of DSF for 2 h (n = 2). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (C)


D Western blot analysis of the phosphorylation level of S6 and p70S6K in WT naïve CD8 + T cells stimulated by α - mouse CD3ε ( 145 - 2C11 ) ( 1 µgml - 1 ) in the presence of α - mouse CD28 ( 1 µgml - 1 ) for the indicated times after pretreatment with DSF ( 5 µM ) for 2 h . The immunoblot shows normalized expression of p - S6 ( S235 / 236 ) to S6 , p - p70S6K to p70S6K , p - Zap70 to Zap70 , as well as that of p - LCK ( Y394 ) and P - Lck ( Y505 ) to Lck . Data information : , data are representative of 3 independent experiments , , Image J analysis in ( D )

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

D Western blot analysis of the phosphorylation level of S6 and p70S6K in WT naïve CD8+ T cells stimulated by α-mouse CD3ε(145-2C11) (1 µgml-1) in the presence of α-mouse CD28 (1 µgml-1) for the indicated times after pretreatment with DSF (5 µM) for 2 h. The immunoblot shows normalized expression of p-S6(S235/236) to S6, p-p70S6K to p70S6K, p-Zap70 to Zap70, as well as that of p-LCK(Y394) and P-Lck(Y505) to Lck. Data information: , data are representative of 3 independent experiments, , Image J analysis in (D)


E FACS analysis of p - CD3ζ ( PY142 ) in mouse naïve CD8 + T cells treated with DSF or stimulated with α - mouse CD3ε ( 145 - 2C11 ) in the presence of α - mouse CD28 ( 1 µgml - 1 ) for 2 h ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; ns , not significant [ unpaired t tests for measurements between the two groups in ( E )

O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-ORGANISM O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FACS analysis of p-CD3ζ(PY142) in mouse naïve CD8+ T cells treated with DSF or stimulated with α-mouse CD3ε(145-2C11) in the presence of α-mouse CD28 (1 µgml-1) for 2 h (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (E)


F Western blot analysis of the indicated proteins in Jurkat T cells treated with the indicated concentrations of DSF for 2 h . The immunoblot analysis shows normalized expression of p - CD3ζ to CD3ζ as well as that of p - Zap70 ( Tyr319 ) / Syk ( Tyr352 ) , p - LCK ( Y394 ) , and p - LAT ( Tyr191 ) to β - actin . Data information : data are representative of 3 independent experiments , Image J analysis in ( F )

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

F Western blot analysis of the indicated proteins in Jurkat T cells treated with the indicated concentrations of DSF for 2 h. The immunoblot analysis shows normalized expression of p-CD3ζ to CD3ζ as well as that of p-Zap70(Tyr319)/Syk(Tyr352), p-LCK(Y394), and p-LAT(Tyr191) to β-actin. Data information: data are representative of 3 independent experiments, Image J analysis in (F)


G , H Luciferin assay of NFAT activity ( G ) and Nfatc1 mRNA expression ( H ) in J76 - NFATRE - luc and TCR052 cells stimulated with α - human CD3 ( OKT3 ) ( 1 µgml - 1 ) in the presence of α - human CD28 ( 1 µgml - 1 ) for 4 h or treated with DSF ( 5 µM ) for 2 h . The levels of NFAT activity were normalized with TCR052 cells treated with DMSO ( n = 3 ) . Data information : data are representative of 3 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; two - way analysis of variance ( ANOVA ) in ( G , H ) ] .

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

G, H Luciferin assay of NFAT activity (G) and Nfatc1 mRNA expression (H) in J76-NFATRE-luc and TCR052 cells stimulated with α-human CD3 (OKT3) (1 µgml-1) in the presence of α-human CD28 (1 µgml-1) for 4 h or treated with DSF (5 µM) for 2 h. The levels of NFAT activity were normalized with TCR052 cells treated with DMSO (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; two-way analysis of variance (ANOVA) in (G, H)].


A , B MST analysis determined the Kd of DSF towards His - LCK ( WT ) ( 86 . 812 nm ) ( A ) or His - LCK ( C20 / 23S ) ( 22 . 797 µM ) ( B ) labeled with RED - tris - NTA 2nd Generation dye . Concentration is reported in nanomolar . Data information : , data are representative of 3 ( A , B , independent

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

A, B MST analysis determined the Kd of DSF towards His-LCK(WT) (86.812 nm) (A) or His-LCK(C20/23S) (22.797 µM) (B) labeled with RED-tris-NTA 2nd Generation dye. Concentration is reported in nanomolar. Data information: , data are representative of 3 (A, B, independent experiments


C , D MS / MS spectra of the Cys20 / 23 - containing human LCK peptide WMENIDVCENCHYPIVPLDGK ( aa 13 - 33 ; 2532 . 87 Da ) ( LC retention time was 49 . 16 min ; a triplet - charged precursor ion m / z 844 . 7163 ( mass , 2531 . 1284 Da ; ΔM of − 0 . 5 ppm ; 10logP = 200 ) was observed ) ( C ) , or of the corresponding LCK peptide after LCK incubation with DSF , which was modified on Cys23 ( red ) by the diethyldithiocarbamate moiety of DSF ( an increase of 147 . 0176 Da ; LC retention time was 56 . 31 min ; a triplet charged precursor ion m / z 893 . 7295 ( mass , 2678 . 1460 Da ; ΔM of 7 . 7 ppm ; − 10logP = 116 . 99 ) was observed ( D ) . Data information : , data are representative of 2 independent

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

C, D MS/MS spectra of the Cys20/23-containing human LCK peptide WMENIDVCENCHYPIVPLDGK (aa 13-33; 2532.87 Da) (LC retention time was 49.16 min; a triplet-charged precursor ion m/z 844.7163 (mass, 2531.1284 Da; ΔM of −0.5 ppm; 10logP = 200) was observed) (C), or of the corresponding LCK peptide after LCK incubation with DSF, which was modified on Cys23 (red) by the diethyldithiocarbamate moiety of DSF (an increase of 147.0176 Da; LC retention time was 56.31 min; a triplet charged precursor ion m/z 893.7295 (mass, 2678.1460 Da; ΔM of 7.7 ppm; −10logP = 116.99) was observed (D). Data information: , data are representative of 2 independent experiments


E Recombinant human LCK proteins left untreated ( Ctrl ) or incubated with ATP and increasing concentrations of DSF for 15 min . Enzyme - linked immunosorbent assay ( ELISA ) of LCK kinase activity was analyzed by using α - p - LCK ( Y394 ) antibody . The results show the average values of the optical density at 450 nm ( OD450 ) from five replicates ( n = 5 ) . Data information : data are representative of 2 independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; ns , not significant [ one - way ANOVA in ( E )

O O B-ORGANISM B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Recombinant human LCK proteins left untreated (Ctrl) or incubated with ATP and increasing concentrations of DSF for 15 min. Enzyme-linked immunosorbent assay (ELISA) of LCK kinase activity was analyzed by using α-p-LCK (Y394) antibody. The results show the average values of the optical density at 450 nm (OD450) from five replicates (n = 5). Data information: data are representative of 2 independent experiments, mean ± SD. **P<0. 01; ***P<0.001; ns, not significant [one-way ANOVA in (E)


F Confocal analysis of p - Lck ( Y394 ) ( red ) in naïve CD8 + T cells treated with DSF ( 5 µM ) or stimulated with α - mouse CD3 and α - mouse CD28 for 2 h ( left ) . The total fluorescence intensity ( IntDen ) was quantified ( right ; n = 20 cells ) . Scale bars , 3 µm . Data information : data are representative of F , independent experiments , mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; ns , not significant [ one - way ANOVA in ( F ) ] .

O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Confocal analysis of p-Lck(Y394) (red) in naïve CD8+ T cells treated with DSF (5 µM) or stimulated with α-mouse CD3 and α-mouse CD28 for 2 h (left). The total fluorescence intensity (IntDen) was quantified (right; n= 20 cells). Scale bars, 3 µm. Data information: data are representative of F, independent experiments, mean ± SD. **P<0. 01; ***P<0.001; ns, not significant [one-way ANOVA in (F)].


G Western blot analysis of the active LCK enrichment [ assessed by p - Lck ( Y394 ) ] in Triton X - 100 - insoluble fractions after DSF ( 5 µM ) treatment in naïve CD8 + T cells . Data information : data are representative of independent experiments ,

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

G Western blot analysis of the active LCK enrichment [assessed by p-Lck (Y394)] in Triton X-100-insoluble fractions after DSF (5 µM) treatment in naïve CD8+ T cells. Data information: data are representative of independent experiments,


H IVP assay of His - tagged CD3εCD peptides was phosphorylated by LCK in the presence of DSF . CD3ε phosphorylation was analyzed by immunoblotting using α - pTyr ( PY100 ) . Data information : , data are representative of 2 , H ) independent

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

H IVP assay of His-tagged CD3εCD peptides was phosphorylated by LCK in the presence of DSF. CD3ε phosphorylation was analyzed by immunoblotting using α-pTyr(PY100). Data information: , data are representative of 2 , H) independent experiments


I Western blot analysis of p - CD3ζ , LCK and p - Zap70 in LCK - deficient cells were infected with the lentivirus collected from human HEK293T cells transfected with control plasmid ( Control ) ( 1 µg ) , LCK ( WT ) ( 1 µg ) , or LCK ( C20 / 23S ) ( 1 µg ) with psPAX2 ( 1 µg ) and pMD2 . G ( 1 µg ) for 48 h followed by treatment with DSF ( 5 µM ) for 2 h stimulated with α - human CD3 and α - human CD28 for 5 min . Data information : data are representative of I ) independent

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

I Western blot analysis of p-CD3ζ, LCK and p-Zap70 in LCK-deficient cells were infected with the lentivirus collected from human HEK293T cells transfected with control plasmid (Control) (1 µg), LCK(WT) (1 µg), or LCK(C20/23S) (1 µg) with psPAX2 (1 µg) and pMD2.G (1 µg) for 48 h followed by treatment with DSF (5 µM) for 2 h stimulated with α-human CD3 and α-human CD28 for 5 min. Data information: data are representative of I) independent experiments


C Scatter plot comparing global gene - expression profiles of DMSO and DSF ( n = 3 ) . Data information : data are representative of 3 independent

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

C Scatter plot comparing global gene-expression profiles of DMSO and DSF (n = 3). Data information: data are representative of 3 independent experiments


D A heatmap of upregulated genes associated with TCR and T cell activation - related genes in DSF relative to that in DMSO . One column indicates one sample in each condition ( n = 3 ) . Data information : data are representative of 3 independent

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

D A heatmap of upregulated genes associated with TCR and T cell activation-related genes in DSF relative to that in DMSO. One column indicates one sample in each condition (n = 3). Data information: data are representative of 3 independent experiments


E ELISA analysis of the production of IL - 2 in primary OT - I T cell supernatants treated with DMSO , DSF ( 5 µM ) or OVA peptide ( 5 µgml - 1 ) for 4 h ( 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 [ unpaired t tests for the measurements between the two groups in ( E )

O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 ELISA analysis of the production of IL-2 in primary OT-I T cell supernatants treated with DMSO, DSF (5 µM) or OVA peptide (5 µgml-1) for 4 h (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 [unpaired t tests for the measurements between the two groups in (E)


F CFSE dilution of proliferation of primary OT - I T cells treated with DMSO or DSF ( 5 µM ) for 2 h ( 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 [ unpaired t tests for the measurements between the two groups in ( F )

O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CFSE dilution of proliferation of primary OT-I T cells treated with DMSO or DSF (5 µM) for 2 h (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 [unpaired t tests for the measurements between the two groups in (F)


G FACS analysis of IFNγ and TNFα production in primary OT - I T cells primed by N4 ( SIINFEKL , 0 . 01 μgml - 1 ) and A4 ( SAINFEKL , 0 . 01 μgml - 1 ) for 2 h followed by treatment with DSF ( 5 µM ) for 2 h ( 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 . two - way ANOVA in ( G )

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

G FACS analysis of IFNγ and TNFα production in primary OT-I T cells primed by N4 (SIINFEKL, 0.01 μgml-1) and A4 (SAINFEKL, 0.01 μgml-1) for 2 h followed by treatment with DSF (5 µM) for 2 h (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. two-way ANOVA in (G)


H , I Extracellular acidification rate ( ECAR ) ( H ) and oxygen consumption rate ( OCR ) ( I ) in 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 . 001 ; * * * * P < 0 . 0001 . ns , not significant [ unpaired t tests for the measurements between the two groups in ( H ) , two - way ANOVA in ( I ) ] .

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

H, I Extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I) in 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<0.001; ****P<0.0001. ns, not significant [unpaired t tests for the measurements between the two groups in (H), two-way ANOVA in (I)].


A , Tumor growth [ ( A ) , ( Vehicle , n = 5 ; DSF , n = 4 , one tumor had disappeared ) ] ( n = 10 mice per group ) ] in DSF ( suspended in 0 . 5 % NaCMC ) - and Vehicle ( equal volume of 0 . 5 % NaCMC ) - treated mice after B16F10 melanoma inoculation . Data information : , data are representative of 3 independent experiments , ns , not significant two - way ANOVA in ( A )

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

A, Tumor growth [(A), (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared)] (n = 10 mice per group)] in DSF (suspended in 0.5% NaCMC)- and Vehicle (equal volume of 0.5% NaCMC)-treated mice after B16F10 melanoma inoculation. Data information: , data are representative of 3 independent experiments, ns, not significant two-way ANOVA in (A)


B Images and tumor weight of subcutaneous mouse B16F10 tumors extracted from mice at day 21 ( Vehicle , n = 5 ; DSF , n = 4 , one tumor had disappeared ) . Data information : , data are representative of 3 independent experiments , ns , not significant [ multiple t tests between the two groups in ( B )

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

B Images and tumor weight of subcutaneous mouse B16F10 tumors extracted from mice at day 21 (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Data information: , data are representative of 3 independent experiments, ns, not significant [multiple t tests between the two groups in (B)


survival curves [ ( C ) , ( n = 10 mice per group ) ] in DSF ( suspended in 0 . 5 % NaCMC ) - and Vehicle ( equal volume of 0 . 5 % NaCMC ) - treated mice after B16F10 melanoma inoculation . Data information : data are representative of 3 independent experiments , mean ± SD . * ns , not significant [ , log - rank ( Mantel - Cox ) test in ( C )

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

survival curves [(C), (n = 10 mice per group)] in DSF (suspended in 0.5% NaCMC)- and Vehicle (equal volume of 0.5% NaCMC)-treated mice after B16F10 melanoma inoculation. Data information: data are representative of 3 independent experiments, mean ± SD. * ns, not significant [ , log-rank (Mantel-Cox) test in (C)


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 )

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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 ) 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 .

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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 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 .

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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 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 )

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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 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 ) ] .

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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 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 )

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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 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

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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 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 ] .

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

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 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 ) .

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

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

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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 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 ) .

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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 ) 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 .

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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 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 .

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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 ) and Atg5flox / flox ; nestin - Cre mice at six weeks of age . 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 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 ) .

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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 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 .

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

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 ) and Atg5flox / flox ; nestin - Cre ( Atg5flox / flox ) mice at six weeks of age , 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 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 .

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

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 + / + 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 .

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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 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 .

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

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 ) and Atg5flox / flox ; CAG - Cre ( Atg5flox / flox ) mice at four months of age , 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 the 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 , and the number of cells with diffuse cytosolic ubiquitin signals , with ( black ) or without ( white ) 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 , and MAVS KO THP1 cells Data information : Panel ( B ) is representative of two independent biological experiments .

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

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


( 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 .

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

(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 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

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

(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 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

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

(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 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 .

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

(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 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

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

(G) 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 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 .

O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM B-GENEPROD O B-ORGANISM B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 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 ) .

O O O B-CELL_LINE O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 ) 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 .

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

(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 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 .

O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM B-GENEPROD O B-GENEPROD O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY 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

(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 . 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 .

O O O B-CELL_LINE B-CELL_TYPE O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-EXP_ASSAY 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

(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 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 .

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

(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 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 .

O O O B-CELL_LINE O O O O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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

(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 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 .

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

(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 . 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 .

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

(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 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 .

O O O B-CELL_LINE O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY 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

(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 , 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(C) 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 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 .

O O O B-CELL_LINE O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD 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

(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 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 ) .

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

(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 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 ) .

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

(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 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 .

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

A-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.