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

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

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(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 obtained in D . mel . S2 by our Luciferase pipeline vs mNeonGreen reporter expression in living D . mel . , revealing a high correlation ( Pearson coefficient 0 . 91 ) between two datasets . Error bars represent standard deviations of 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

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

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

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

O O O O O O O O O O O O O O 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) 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 grouped by native promoter sequences . colors refer to different promoter architectures . Three types of are used to indicate expression fold change with no increase ( y = x ; solid ) , 2 - fold increase ( y = x + 1 ; dotted ) 4 - fold increase ( y = x + 2 ; dashed ) . Red vertical dashed : log2 basal expressions = 2 .

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

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

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

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

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 of relative of S - nitrosylated IRE1 ( f ) total IRE1 ( g ) in brains from young aged mice . Histograms show relative of SNO - IRE1 / input IRE1α measured by biotin - switch 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 ) . Data information :

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (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 ) aged ( 18 - 20 months ) conditional knockout for IRE1 ( IRE1cKO ) their flox / flox control ( IRE1WT ) were evaluated ( D ) Mean of of β - galactosidase in CA3 region ( n = 3 - 5 / 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 O O O O O O O O O O O O O O O O O O O O O O O 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 of dendritic spines ( arrows ) in CA1 region of middle - aged IRE1WT injected with AAV - Mock or AAV - Cre . 60x magnification , ( G ) Mean of spine density per μm ( n = 3 , 3 ; 4 , 4 ) . P computed by unpaired Student ' s t within each - matched 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 O O O O O O O O O O O O O O O O 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 of β - galactosidase of hippocampal slices derived from aged ( 22 - 24 months ) TgXBP1s WT . ( F ) Mean of of stained by β - galactosidase in hippocampus of aged ( 20 - 24 months ) TgXBP1s compared to littermate controls .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 s . e . m . of of γH2A - positive ; unpaired Student ' s t comparing aged groups ( n = 6 , 6 / group ; unpaired Student ' s t ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of p - S6 in WT mouse splenic CD8 + T 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 for measurements between two groups in ( B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 stimulated for 8 min with α - mouse CD3ε ( 145 - 2C11 ) after pretreatment with 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 for measurements between two groups in ( C )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of phosphorylation level of S6 p70S6K in WT naïve CD8 + T stimulated by α - mouse CD3ε ( 145 - 2C11 ) ( 1 µgml - 1 ) in presence of α - mouse CD28 ( 1 µgml - 1 ) for indicated times after pretreatment with DSF ( 5 µM ) for 2 h . 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 ) P - Lck ( Y505 ) to Lck . Data information : , data are representative of 3 independent experiments , , Image J in ( D )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 of p - CD3ζ ( PY142 ) in mouse naïve CD8 + T treated with DSF or stimulated with α - mouse CD3ε ( 145 - 2C11 ) in 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 for measurements between two groups in ( E )

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

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 of NFAT activity ( G ) Nfatc1 mRNA expression ( H ) in J76 - NFATRE - luc TCR052 stimulated with α - human CD3 ( OKT3 ) ( 1 µgml - 1 ) in presence of α - human CD28 ( 1 µgml - 1 ) for 4 h or treated with DSF ( 5 µM ) for 2 h . of NFAT activity were normalized with TCR052 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 of variance ( ANOVA ) in ( G , H ) ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 determined 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 . is reported in nanomolar . Data information : , data are representative of 3 ( A , B , independent

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 corresponding LCK peptide after LCK incubation with DSF , which was modified on Cys23 ( red ) by 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 left untreated ( Ctrl ) or incubated with ATP increasing concentrations of DSF for 15 min . Enzyme - linked immunosorbent ( ELISA ) of LCK kinase activity was analyzed by using α - p - LCK ( Y394 ) antibody . results show average values of 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 )

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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 of p - Lck ( Y394 ) ( red ) in naïve CD8 + T treated with DSF ( 5 µM ) or stimulated with α - mouse CD3 α - mouse CD28 for 2 h ( left ) . total ( IntDen ) was quantified ( right ; n = 20 ) . 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 ) ] .

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

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

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

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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 DSF ( n = 3 ) . Data information : data are representative of 3 independent

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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 of upregulated genes associated with TCR T 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

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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 of production of IL - 2 in OT - I T 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 for measurements between two groups in ( E )

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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 OT - I T 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 for measurements between two groups in ( F )

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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 of IFNγ TNFα production in OT - I T primed by N4 ( SIINFEKL , 0 . 01 μgml - 1 ) 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 )

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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 ) oxygen consumption rate ( OCR ) ( I ) in OT - I T ( 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 for measurements between two groups in ( H ) , two - way ANOVA in ( I ) ] .

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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 , growth [ ( A ) , ( Vehicle , n = 5 ; DSF , n = 4 , one had disappeared ) ] ( n = 10 mice per group ) ] in DSF ( suspended in 0 . 5 % NaCMC ) - 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 )

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

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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 ) - 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 ) in ( C )

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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