( C ) Apoptosis in U2OS cotransfected with scrambled or TIGAR siRNAs , scrambled , ATG5 siRNA or ATG10 siRNA , as measured by with a sub - G1 content . After 48 h , were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . Data are shown as mean standard deviation from three independent experiments . In each case , increase in apoptosis after knockdown of ATG5 or ATG10 , compared with matched control , was statistically significant ; * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

(C) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled, ATG5 siRNA or ATG10 siRNA, as measured by cells with a sub-G1 DNA content. After 48 h, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. Data are shown as the mean and standard deviation from three independent experiments. In each case, the increase in apoptosis after knockdown of ATG5 or ATG10, compared with the matched control, was statistically significant; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


( G ) Liver were resolved by PAGE binding of serum antibodies from 4 mice from each group ( numbered 1 - 4 ) was detected by anti - IgG secondary antibody .

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(G) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group (numbered 1-4) was detected by anti-IgG secondary antibody .


C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . After 2 , 7 μm spleen sections were stained with Red : Alexa - Fluor - 594 - CD4 Green : FITC - B220 ( D ) or FITC - GL7 ( E ) . Scale bar represent 100 nm .

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Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. After 2 weeks, 7 μm spleen sections were stained with Red:Alexa-Fluor-594-CD4 and Green: FITC-B220 (D) or FITC-GL7 (E). Scale bar represent 100 nm.


C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . Serum was assessed for level of IgG antibodies binding NP - 31 NP - 4 ( F ) .

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Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. Serum was assessed for level of IgG antibodies binding NP-31 and NP-4 (F).


( A ) PBMC were isolated from healthy volunteers stained for flow cytometry to identify NRP1 expression in CD3 + CD4 + Th against CD127 ( left ) intracellular Foxp3 ( right )

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(A) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3+CD4+ Th cells against CD127 (left) and intracellular Foxp3 (right)


NRP1 - expressing amongst CD4 + CD3 + Th were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) 10 SLE . Representative is shown for CD4 + CD3 + Th ( B ) , summary data for CD3 + CD4 + Th ( C ) Plots show subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T .

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NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. Representative staining is shown for CD4+CD3+ Th cells (B), and summary data for CD3+CD4+ Th cells (C) Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.


NRP1 - expressing amongst CD4 + CD3 + Th were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) 10 SLE . summary data for CD3 + CD4 + CD127hi conventional ( D ) . Plots show subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T .

B-MEASURED_VAR O O O B-MEASURED_VAR 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 B-MEASURED_VAR O B-MEASURED_VAR 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

NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. summary data for CD3+CD4+CD127hi conventional cells (D). Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.


( E ) Individuals were divided into NRP1 - high subjects ( NRP1 + > 1 . 3 % in Th ) NRP1 - low subjects ( NRP1 + ≤ 1 . 35 % in Th ) . Numbers of HC SLE falling into each group are shown . * * * p < 0 . 001 Chi squared with Yates ' correction .

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(E) Individuals were divided into NRP1-high subjects (NRP1+ cells >1.3% in Th cells) and NRP1-low subjects (NRP1+ cells≤1.35% in Th cells). Numbers of HC and SLE falling into each group are shown. *** p<0.001 Chi squared test with Yates' correction.


( F , G ) NRP1 - expressing amongst CD3 + CD4 + CD45RO - were measured , representative ( F ) summary data ( G ) . plot shows subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * * p < 0 . 0001 Unpaired Student ' s T .

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(F, G) NRP1-expressing cells amongst CD3+CD4+CD45RO- cells were measured, representative staining (F) and summary data (G). The plot shows individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ****p<0.0001 Unpaired Student's T test.


B . Bright - field picture of a 0B K8 - eYFP ( Left ) time series of two - photon live imaging of ExE region ( Right ) . Time interval is 5 min ( n = 3 ) . C . Bright - field picture of EB K8 - eYFP ( Left ) time series of two - photon live imaging of ExE region ( Right ) . Time interval is 5 min ( n = 4 ) . D . Bright - field pictures of K8 - eYFP LB ( n = 30 ) , high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed areas . K8 - eYFP DAPI appear in yellow cyan , respectively .

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B. Bright-field picture of a 0B K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=3). C. Bright-field picture of EB K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=4). D. Bright-field pictures of K8-eYFP LB embryos (n=30), and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas. K8-eYFP and DAPI appear in yellow and cyan, respectively. Da


A - H . of an opened ( A ) , ExE cavity wall ( B ) where blood islands are indicated by arrow , allantois surface ( C - C ' ) , an opened allantois ( D ) its zooms ( E - E ' ) where filaments are indicated by arrows , epiblast - derived side of amnion ( F ) , mesoderm - derived side of amnion ( G ) its zoom ( G ' ) .

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A-H. SEM images of an opened embryo (A), the ExE cavity wall (B) where blood islands are indicated by white arrow, the allantois surface (C-C'), an opened allantois (D) and its zooms (E-E') where filaments are indicated by white arrows, the epiblast-derived side of the amnion (F), the mesoderm-derived side of the amnion (G) and its zoom (G').


B . Sagittal sections of C57BL / 6J wild - type KtyII - / - E7 . 5 immunostained for Krt8 ( n = 15 n = 5 , respectively ) , F - actin ( n = 11 2 ) , Vimentin ( n = 5 1 ) , E - cadherin ( n = 1 1 ) , T ( n = 4 3 ) , Dsg2 ( n = 15 5 ) Pecam - 1 ( n = 4 3 ) . Data information : arrows point toward allantois scale bars represent 25 µm .

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B. Sagittal sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Krt8 (n= 15 and n=5, respectively), F-actin (n=11 and 2), Vimentin (n=5 and 1), E-cadherin (n=1 and 1), T (n=4 and 3), Dsg2 (n=15 and 5) and Pecam-1 (n=4 and 3). Data information: White arrows point toward allantois and scale bars represent 25 µm.


of at LB stage in C57BL / 6J wild - type ( boxes / black dots , n = 22 including 16 in which allantois could be measured ) , KtyII - / - ( grey boxes / black triangles , n = 9 including 8 in which allantois could be measured ) . ( D ) Embryonic region . Ratios of whole ExE cavity lengths ( E ) , over embryonic region .

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Quantification of embryo morphology at LB stage in C57BL/6J wild-type (white boxes/ black dots, n=22 including 16 in which allantois could be measured), and KtyII-/- embryos (grey boxes/ black triangles, n=9 including 8 in which allantois could be measured). (D) Embryonic region length. Ratios of whole embryo and ExE cavity lengths (E) , over embryonic region length.


I - I ' ' . of opened C57BL / 6J wild - type ( n = 3 ) KtyII - / - ( n = 2 ) LB stage with zooms on allantois region ( I ' - I ' ' ) . Scale bars represent 200 ( I ) , 20 ( I ' ) 10 ( I ' ' ) µm .

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I-I''. SEM images of opened C57BL/6J wild-type (n=3) and KtyII-/- (n=2) LB stage embryos with zooms on the allantois region (I'-I''). Scale bars represent 200 (I), 20 (I') and 10 (I'') µm.


F . Dot plot of specific gene expression in amnion clusters .

F. Dot plot of specific gene expression in amnion clusters.


G . Immunostaining for Cdx2 ( n = 20 ) . H . Immunostaining for Nrp1 ( n = 8 ) Col1a1 ( n = 7 ) , primarily expressed in Amnion Mesenchyme . I . Immunostaining for Epcam ( n = 12 ) , Cldn7 ( n = 7 ) , Lrp2 ( n = 6 ) , primarily expressed in Amnion Ectoderm .

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G. Immunostaining for Cdx2 (n=20). H. Immunostaining for Nrp1 (n=8) and Col1a1 (n=7), primarily expressed in Amnion Mesenchyme. I. Immunostaining for Epcam (n=12), Cldn7 (n=7), and Lrp2 (n=6), primarily expressed in Amnion Ectoderm.


J - K . Sections of C57BL / 6J wild - type KtyII - / - E7 . 5 immunostained for Nrp1 ( K ) ( n = 8 2 , respectively ) , Lrp2 ( L ) ( n = 3 2 ) .

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J-K. Sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Nrp1 (K) (n=8 and 2, respectively), and Lrp2 (L) (n=3 and 2).


D . Dot plot of specific gene expression in chorion clusters .

D. Dot plot of specific gene expression in chorion clusters.


E . Immunostaining for Cdx2 ( n = 20 ) . F . Immunostaining for DAPI ( cyan , left ) , Sox2 ( middle ) , Gata2 ( right ) . Sox2 is primarily expressed in Trophoblast Progenitors Gata2 in Chorion Ectoderm . G . Immunostaining for Cebpb , detectable in most with variable . H . Immunostaining for Fn1 ( n = 22 ) , Dlk1 ( n = 8 ) , Acta2 ( n = 12 ) , primarily expressed in Chorion Mesenchyme . I . Sections from C57BL / 6J wild - type KtyII - / - LB immunostained for Dlk - 1 ( n = 8 4 , respectively ) Acta2 ( n = 6 3 ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O

E. Immunostaining for Cdx2 (n=20). F. Immunostaining for DAPI (cyan, left), Sox2 (middle), and Gata2 (right). Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm. G. Immunostaining for Cebpb, detectable in most cells with variable intensity. H. Immunostaining for Fn1 (n=22), Dlk1 (n=8), and Acta2 (n=12), primarily expressed in Chorion Mesenchyme. I. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Dlk-1 (n=8 and 4, respectively) and Acta2 (n=6 and 3). Data


D . Dot plot of specific gene expression in allantois clusters .

D. Dot plot of specific gene expression in allantois clusters.


F . Immunostaining for Ifitm3 ( n = 7 ) ( Allantois Base ) . G . Immunostaining for Cdh11 ( n = 9 ) , Stard8 ( n = 4 ) Nrp1 ( n = 8 ) ( Allantois Cortex ) . H . Immunostaining for Pitx1 ( n = 4 ) ( Allantois Mesenchyme ) . I . Hhex - GFP ( n = 5 ) immunostaining for Kdr ( n = 8 ) ( Endothelial Progenitors ) . J . Immunostaining for Runx1 ( n = 3 ) ( Erythroid Progenitors ) . K - L .

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F. Immunostaining for Ifitm3 (n=7) (Allantois Base). G. Immunostaining for Cdh11 (n=9), Stard8 (n=4) and Nrp1 (n=8) (Allantois Cortex). H. Immunostaining for Pitx1 (n=4) (Allantois Mesenchyme). I. Hhex-GFP (n=5) and immunostaining for Kdr (n=8) (Endothelial Progenitors). J. Immunostaining for Runx1 (n=3) (Erythroid Progenitors). K-L.


K - L . Sections from C57BL / 6J wild - type KtyII - / - LB immunostained for Cdh11 ( Top ) Krt8 ( Bottom ) ( K , n = 8 4 , respectively ) Ifitm3 ( L , n = 2 2 ) . Data information : arrows point toward signal in allantois . Sagittal sections with anterior to left .

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K-L. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Cdh11 (Top) and Krt8 (Bottom) (K, n=8 and 4, respectively) and Ifitm3 (L, n=2 and 2). Data information: White arrows point toward signal in allantois. Sagittal sections with anterior to the left.


( A ) Srcap expression in murine different tissues was examined by Northern blot . A 277 nt probe of Srcap ( 81 - 358nt ) was labeled for Northern blot . 18S was used as a loading control .

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(A) Srcap expression in murine different tissues was examined by Northern blot. A 277 nt probe of Srcap (81-358nt) was labeled for Northern blot analysis. 18S RNA was used as a loading control.


( B ) Two - week - old SRCAP - C - HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence . A global look of section was shown . Scale bar : 500 μm . Green : EpCAM , Red : HA tag , Nuclei were counterstained by DAPI .

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(B) Two-week-old SRCAP-C-HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining. A global look of the section was shown. Scale bar: 500 μm. Green: EpCAM, Red: HA tag, Nuclei were counterstained by DAPI.


( C ) Murine intestine tissues were fixed stained according to iDISCO protocol . SRCAP was highly expressed in vast majority of crypts in mouse intestine . Scale bar : 50 μm . Green : EpCAM , Red : SRCAP , Nuclei were counterstained by DAPI .

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(C) Murine intestine tissues were fixed and stained according to iDISCO staining protocol. SRCAP was highly expressed in vast majority of crypts in mouse intestine. Scale bar: 50 μm. Green: EpCAM, Red: SRCAP, Nuclei were counterstained by DAPI.


( D ) Duodenum region of intestinal tissues were obtained from Lgr5GFP - CreERT2 ( Lgr5GFP ) mice for immunofluorescence by indicated antibodies with OpalTM 7 - color fIHC kit . Nuclei were counterstained with DAPI . Scale bar : 50 μm .

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(D) Duodenum region of intestinal tissues were obtained from Lgr5GFP-CreERT2 (Lgr5GFP) mice for immunofluorescence staining by indicated antibodies with OpalTM 7-color fIHC kit. Nuclei were counterstained with DAPI. Scale bar: 50 μm.


( E ) ISCs from Lgr5GFP mice were collected for organoid formation . Organoids were fixed for immunofluorescence with indicated antibodies . Scale bar : 50 μm . Data information : Green : EpCAM , Red : SRCAP , Purple : PCNA , Nuclei were counterstained by DAPI .

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(E) ISCs from Lgr5GFP mice were collected for organoid formation. Organoids were fixed for immunofluorescence staining with indicated antibodies. Scale bar: 50 μm. Data information: Green: EpCAM, Red: SRCAP, Purple: PCNA, Nuclei were counterstained by DAPI.


( A ) Representative of intestines from 8 - week - old Srcap + / + Srcap - / - mice ( n = 6 per group ) . of 120 villi 120 crypts ( 20 villi 20 crypts per mouse ) was measured shown in right panel as means ± S . D . * * P < 0 . 01 by two - Student ' s t - . Scale bar , 100 μm . Dotted lines indicate boundary between crypts villi . Arrows indicate of crypts or villi .

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(A) Representative images of intestines from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) was measured and shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.


( B ) Intestinal tissues from 8 - week - old Srcap + / + Srcap - / - mice ( n = 6 per group ) were collected for immunofluorescence with indicated antibodies . Typical were shown in left panel statistical ISC numbers from 120 crypts ( 20 crypts per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - Student ' s t - . Scale bar , 50 μm .

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(B) Intestinal tissues from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group) were collected for immunofluorescence staining with indicated antibodies. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.


( C ) 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 3 for each group . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - Student ' s t - .

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(C) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.


( D ) Srcap + / + Srcap - / - mice were treated with 10 Gy ' s radiation sacrificed at different time points . Intestinal tissues were collected for H & E . Numbers of intact crypts were shown in lower panel as means ± S . D . * * P < 0 . 01 by two - Student ' s t - . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) . Scale bars , 100 μm .

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(D) Srcap+/+ and Srcap-/- mice were treated with 10 Gy's radiation and sacrificed at different time points. Intestinal tissues were collected for H&E staining. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 100 μm.


( E ) Lgr5GFP - CreERT2 ; Rosa26lsl - YFP ( LRYFP ) were crossed with Srcapflox / flox mice , followed by administration of tamoxifen ( TAM ) for lineage tracing . Mice were sacrificed 7 days after TAM induction typical jejunum sections were stained . Numbers of traced crypt - villus units were shown in right panel as means ± S . D . * * P < 0 . 01 by two - Student ' s t - . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) . Scale bars , 50 μm .

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(E) Lgr5GFP-CreERT2;Rosa26lsl-YFP (LRYFP) were crossed with Srcapflox/flox mice, followed by administration of tamoxifen (TAM) for lineage tracing analysis. Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained. Numbers of traced crypt-villus units were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 50 μm.


( F ) Proliferation of Srcap + / + Srcap - / - ISCs were examined by Ki67 followed by flow cytometry . Percentages of proliferated were counted as means ± S . D ( lower panel ) . * * P < 0 . 01 by two - Student ' s t - . n = 5 per group .

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(F) Proliferation of Srcap+/+ and Srcap-/- ISCs were examined by Ki67 staining followed by flow cytometry analysis. Percentages of proliferated cells were counted as means ± S.D (lower panel). ** P < 0.01 by two-tailed Student's t-test. n=5 per group.


( G ) Multiple color was performed in Srcap + / + Srcap - / - intestine sections . AP - Red , DAB AP - Blue were used for immunohistochemistray coloration . Numbers of each kind of epithelial from 50 crypts or 50 villi ( 10 crypts 10 villi per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - Student ' s t - . Scale bars , 50 μm .

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(G) Multiple color staining was performed in Srcap+/+ and Srcap-/- intestine sections. AP-Red, DAB and AP-Blue were used for immunohistochemistray coloration. Numbers of each kind of epithelial cells from 50 crypts or 50 villi (10 crypts and 10 villi per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bars, 50 μm.


( A ) extracts of murine intestinal crypts were immunoprecipitated with anti - SRCAP antibody or rabbit IgG followed by ADASDS - PAGE , sliver mass spectrometry .

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(A) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti-SRCAP antibody or rabbit IgG followed by ADASDS-PAGE, sliver staining and mass spectrometry.


( B ) SRCAP were stained in murine intestinal crypts . Scale bar , 10 μm .

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(B) SRCAP and REST were stained in murine intestinal crypts. Scale bar, 10 μm.


( D ) Flag - tagged fragments were expressed in 293T purified by immunoprecipitation with anti - Flag antibody . were incubated with crypt lysates for pulldown followed by immunoblotting .

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(D) Flag-tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti-Flag antibody. REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting.


( E ) C - terminal domain of was sufficient essential for SRCAP association by pulldown .

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(E) C-terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay.


( F ) Intestinal crypt lysates were incubated with anti - antibody , followed by immunoblotting with indicated antibodies .

(F) Intestinal crypt lysates were incubated with anti-REST antibody, followed by immunoblotting with indicated antibodies.


( G ) Intestinal crypt lysates were incubated with anti - antibody A / G beads in 4 ℃ overnight . Recombinant SRCAP was added with different concentrations , followed by immunoblotting .

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(G) Intestinal crypt lysates were incubated with anti-REST antibody and protein A/G beads in 4℃ overnight. Recombinant SRCAP protein was added with different concentrations, followed by immunoblotting.


( B ) Transcriptome microarray between Srcap - / - ISCs WT ISCs . Top ten downregulated TFs in Srcap - / - ISCs were listed .

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(B) Transcriptome microarray analysis between Srcap-/- ISCs and WT ISCs. Top ten downregulated TFs in Srcap-/- ISCs were listed.


( C ) Prdm16 expression was tested in Srcap + / + Srcap - / - ISCs by Western blotting .

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(C) Prdm16 expression was tested in Srcap+/+ and Srcap-/- ISCs by Western blotting.


( D ) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence . Green : GFP ; Red : SRCAP ; Blue : Prdm16 . Scale bar , 10 μm .

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(D) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining. Green: GFP; Red: SRCAP; Blue: Prdm16. Scale bar, 10 μm.


( E ) Prdm16 promoter ( - 875 ~ - 854 ) region was used as probe to perform - pulldown with murine intestinal crypt lysates followed by immunoblotting .

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(E) Prdm16 promoter (-875~-854) region was used as probe to perform DNA-pulldown assay with murine intestinal crypt lysates followed by immunoblotting.


( F , G ) Indicated promoter activation was analyzed with luciferase . DKK1 was used as a control . Results of relative fold changes are shown as means ± S . D . Data represent five independent replicates . . * * p < 0 . 01 by two - Student ' s t - . .

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(F, G) Indicated promoter activation was analyzed with luciferase assay. DKK1 was used as a control. Results of relative fold changes are shown as means ± S.D. Data represent five independent replicates. . ** p<0.01 by two-tailed Student's t-test. .


( H ) 1 × 104 ISCs from indicated mice were collected for organoid formation . For gene depletion or restoration , ISCs were infected with lentivirus . Scale bar , 200 μm . Data information : data represent five independent experiments .

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(H) 1×104 ISCs from indicated mice were collected for organoid formation. For gene depletion or restoration, ISCs were infected with lentivirus. Scale bar, 200 μm. Data information: All data represent five independent experiments.


( A ) WT Srcap - / - ISCs were conducted for transcriptome microarray , followed by Gene Set Enrichment ( GSEA ) for PPARδ target genes .

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(A) WT and Srcap-/- ISCs were conducted for transcriptome microarray, followed by Gene Set Enrichment Analysis (GSEA) analysis for PPARδ target genes.


( B ) Expression of PPARs PPAR target genes were assessed in sorted ISCs by quantitative RT - PCR . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin shown as means ± S . D . Data represent five independent replicates . * * p < 0 . 01 * * * p < 0 . 001 by two - Student ' s t - .

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(B) Expression levels of PPARs and PPAR target genes were assessed in sorted ISCs by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 and *** p<0.001 by two-tailed Student's t-test.


( C ) PPARδ expression was visualized in intestinal tissues by immunofluorescence . Scale bar , 50 μm .

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(C) PPARδ expression was visualized in intestinal tissues by immunofluorescence staining. Scale bar, 50 μm.


( D ) ISCs were collected for organoid formation followed by quantitative RT - PCR . For Prdm16 overexpression , ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin shown as means ± S . D . Data represent five independent replicates . * * p < 0 . 01 by two - Student ' s t - .

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(D) ISCs were collected for organoid formation followed by quantitative RT-PCR. For Prdm16 overexpression, ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 by two-tailed Student's t-test.


( E ) mRNA of ISC signature genes were examined by quantitative RT - PCR . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin shown as means ± S . D . Data represent five independent replicates .

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(E) mRNA levels of ISC signature genes were examined by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates.


( F ) Representative of organoid formation from indicated ISCs were shown ( upper panel ) . Scale bar , 200 μm . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - Student ' s t - . ( lower panel ) . n = 6 for each group . GW501516 ( 10 μM ) were added directly into organoid culture medium .

(F) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test. (lower panel). n=6 for each group. GW501516 (10 μM) were added directly into organoid culture medium.


( A ) Representative of intestines from 8 - week - old + / + - / - mice ( n = 6 per group ) . of 120 villi 120 crypts ( 20 villi 20 crypts per mouse ) were measured in right panel as means ± S . D . * * P < 0 . 01 by two - Student ' s t - . Scale bar , 100 μm . Dotted lines indicate boundary between crypts villi . Arrows indicate of crypts or villi .

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(A) Representative images of intestines from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) were measured in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.


( B ) 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 3 for each group . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - Student ' s t - .

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(B) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.


( C ) Intestine tissues from 8 - week - old + / + - / - mice ( n = 6 per group ) were collected for immunohistochemistry . Typical were shown in left panel statistical ISC numbers from 120 crypts ( 20 crypts per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - Student ' s t - . Scale bar , 50 μm .

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(C) Intestine tissues from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group) were collected for immunohistochemistry staining. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.


( D ) Immunofluorescence of intestine tissues from Srcapfl / fl ; Lgr5GFP - CreERT2 Restfl / fl ; Lgr5GFP - CreERT2 mice . Scale bar , 50 μm .

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(D) Immunofluorescence staining of intestine tissues from Srcapfl/fl; Lgr5GFP-CreERT2 and Restfl/fl; Lgr5GFP-CreERT2 mice. Scale bar, 50 μm.


( E ) Lgr5GFP - CreERT2 ; Rosa26lsl - lacZ ( LRlacZ ) were crossed with - / - mice , followed by TAM administration for lineage tracing for intestinal whole - mount for β - gal ( upper panel ) . Mice were sacrificed after one week typical sections were shown ( middle panel ) . Scale bar , 200 μm . Numbers of traced crypt - villus units were calculated as means ± S . D . * * P < 0 . 01 by two - Student ' s t - in right panel . 50 fields ( 10 fields per mouse ) were calculated for each group ( n = 5 ) ( lower panel ) .

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(E) Lgr5GFP-CreERT2;Rosa26lsl-lacZ (LRlacZ) were crossed with Rest-/- mice, followed by TAM administration for lineage tracing for intestinal whole-mount staining for β-gal (upper panel) . Mice were sacrificed after one week and typical sections were shown (middle panel). Scale bar, 200 μm. Numbers of traced crypt-villus units were calculated as means± S.D. ** P < 0.01 by two-tailed Student's t-test in right panel. 50 fields (10 fields per mouse) were calculated for each group (n=5) (lower panel).


( F ) + / + - / - mice were treated with 10 Gy ' s radiation sacrificed at indicated time points . Intestinal tissues were collected for H & E . Scale bars , 100 μm . Numbers of intact crypts were shown in lower panel as means ± S . D . * * P < 0 . 01 by two - Student ' s t - . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) .

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(F) Rest+/+ and Rest-/- mice were treated with 10 Gy's radiation and sacrificed at the indicated time points. Intestinal tissues were collected for H&E staining. Scale bars, 100 μm. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5).


( G ) Representative of organoid formation from indicated ISCs were shown ( upper panel ) . Scale bar , 200 μm . n = 6 for each group . Organoid number per well was counted as means ± S . D . ( lower panel ) . Lentivirus - carried sgPrdm16 sgPpard were prepared in 293T infected into ISCs derived from TAM - treated LRCas9 mice to obtain Prdm16 - / - Ppard - / - ISCs . 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 5 for each group .

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(G) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. n=6 for each group. Organoid number per well was counted as means ± S.D. (lower panel). Lentivirus-carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM-treated LRCas9 mice to obtain Prdm16-/- and Ppard-/- ISCs. 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=5 for each group.


( B - D ) Immunofluorescent of mouse liver ( B ) , testis ( C ) heart ( D ) . H3K27ac , PolII - S5P , laminin were stained with specific antibodies visualized using labeled anti - mouse ChIL - probe ( red : H3K27ac PolIIS5P ) anti - rabbit IgG ( green : laminin ) . was counterstained with Hoechst 33342 . Scale Bar : 20 µm ( left ) , 10 µm ( right ) .

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(B-D) Immunofluorescent images of mouse liver (B), testis (C) and heart (D). H3K27ac, PolII-S5P, and laminin were stained with specific primary antibodies and visualized using fluorescent labeled anti-mouse ChIL-probe (red: H3K27ac and PolIIS5P) and anti-rabbit IgG (green: laminin). DNA was counterstained with Hoechst 33342. Scale Bar: 20 µm (left images), 10 µm (right images).


( E ) Genome browser of ChIL - seq for H3K27ac PolII - S5P bulk tissue - seq data at Alb locus in liver tissues .

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(E) Genome browser images of ChIL-seq for H3K27ac and PolII-S5P and bulk tissue RNA-seq data at the Alb locus in liver tissues.


( B ) IGV tracks of H3K27ac ChIL - seq at identified tissue - specific enhancers of Rxra , Gnat3 , Eps8 , a house - keeping gene of Actb loci are shown with replicates .

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(B) The IGV tracks of H3K27ac ChIL-seq at identified tissue-specific enhancers of Rxra, Gnat3, Eps8, and a house-keeping gene of Actb loci are shown with the replicates.


( C ) Specific motif enrichment was conducted using chromVAR ( Schep et al , 2017 ) . Hierarchical clustering of deviation - Z scores of three replicates of each tissue is shown .

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(C) Specific motif enrichment analysis was conducted using chromVAR(Schep et al, 2017). Hierarchical clustering of deviation-Z scores of three replicates of each tissue is shown.


( D ) Super - enhancer identification . Tissue - specific enhancers are identified , so that they are listed more than twice ( twice : blue , : red ) in top 5 % in enhancer candidates are not in SEs of other tissues . Grey shades indicate top 5 % of tag in enhancer candidates .

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(D) Super-enhancer identification. Tissue-specific enhancers are identified, so that they are listed more than twice (twice: blue, all: red) in the top 5% in all enhancer candidates and are not in the SEs of other tissues. Grey shades indicate the top 5% of tag count in enhancer candidates.


( F ) Hnf4ɑ binds to SE at Alb gene loci .

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(F) Hnf4ɑ binds to the SE at Alb gene loci.


( B ) Signal intensities of ChIL - seq correlated with expression of genes . lines indicate average CPM of each expression group at TSS . expression groups were assigned with respect to expression ( TPM ) of genes .

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(B) Signal intensities of ChIL-seq correlated with the expression levels of genes. The lines indicate the average CPM of each expression group at TSS. The expression groups were assigned with respect to the expression levels (TPM) of genes.


( E ) tissue - specific genes identified by PolII S5P ChIL - seq . IGV tracks of replicates of PolII S5P ChIL - seq are shown at each specific gene ( Trf , Myh6 , Meig for liver , heart , testis , respectively ) . Actb is also shown as ubiquitously expressed gene in three tissues .

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(E) The tissue-specific genes identified by PolII S5P ChIL-seq. The IGV tracks of all replicates of PolII S5P ChIL-seq are shown at each specific gene (Trf, Myh6, and Meig for the liver, heart, and testis, respectively). Actb is also shown as the ubiquitously expressed gene in the three tissues.


( B ) PolII S5P ChIL - seq signal of marker genes of mature skeletal muscle ( Acta1 ) macrophages ( Cd68 ) .

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(B) PolII S5P ChIL-seq signal of the marker genes of mature skeletal muscle (Acta1) and macrophages (Cd68).


( F ) Bulk - tissue expression ( TPM ) of representative genes .

(F) Bulk-tissue expression levels (TPM) of the representative genes.


( H ) Activities of major types in muscle regeneration . colors of show log2FC to day 0 ( uninjured ) of TR TSS . Representative genes with significant changes in TR are shown .

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(H) Activities of major cell types in muscle regeneration. The colors of the heatmap show the log2FC to day 0 (uninjured) of TR and TSS levels. Representative genes with significant changes in TR are shown.


( A ) IGV screen shots of PolII S5P ChIL signals are shown for representative genes in breast cancer tissues . For each lane , samples were selected from each progressive stage of breast cancer .

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(A) IGV screen shots of PolII S5P ChIL signals are shown for representative genes in breast cancer tissues. For each lane, individual samples were selected from each progressive stage of breast cancer.


( C ) Expression , TSS level , TR of genes . EEF1A1 is shown as a control housekeeping gene . horizontal dotted indicates median level of genes . Error bars indicate 95 % confidence intervals for each estimate indicated by dots ( N = 1 for each ) .

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(C) Expression, TSS level, and TR of genes. EEF1A1 is shown as a control housekeeping gene. The horizontal dotted line indicates the median level of all genes. Error bars indicate 95% confidence intervals for each estimate indicated by dots (N=1 for each).


A . qPCR data comparing expression of CamkIIδc transgene in brain heart of 3 month old CamkIIδc TG mice ; n = 4 / group ; * P < 0 . 05 , Unpaired t - , two - . Note that transgene is not detected in brain .

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A. qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice; n=4/group; *P <0.05, Unpaired t-test, two-tailed. Note that the transgene is not detected in the brain.


C . Ejection fraction , cardiac output index are significantly decreased in CamkIIδc TG mice ( n = 8 ) when compared to control mice ( n = 5 ; * P < 0 . 05 , , * * P < 0 . 01 , * * * P < 0 . 001 , Unpaired t - , two - ) . Heart to , left ventricle to , lung to are increased in CamkIIδc TG ( n = 8 ) compared to control ( n = 5 ; * P < 0 . 05 , * * P < 0 . 01 , Unpaired t - ; two - ) .

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C. Ejection fraction, cardiac output and index are significantly decreased in CamkIIδc TG mice (n =8) when compared to control mice (n=5; *P <0.05, , **P<0.01, ***P<0.001, Unpaired t-test, two-tailed). Heart to body ratio, left ventricle to body ratio, and lung to body weight ratio are increased in CamkIIδc TG (n=8) compared to control (n=5; *P <0.05, **P<0.01, Unpaired t-test; two-tailed).


H . qPCR of selected genes reflecting ER - stress or methylation - related processes in CamkIIδc TG mice ( n = 4 ) control mice ( n = 5 ) . * p < 0 . 05 , ns = non - significant , Unpaired t - ; two - . Data is normalized to Hprt1 expression .

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H. qPCR quantification of selected genes reflecting ER-stress or protein methylation-related processes in CamkIIδc TG mice (n=4) and control mice (n=5). *p<0.05, ns = non-significant, Unpaired t-test; two-tailed. Data is normalized to Hprt1 expression.


D . cumulative score of hippocampus - dependent search strategies during Barnes maze training is impaired in CamkIIδc TG ( n = 16 ) when compared to control mice ( n = 13 ; two - , unpaired t - , * p < 0 . 05 ) . E . Number of visits to escape hole during probe to memory retrieval was impaired in CamkIIδc TG ( n = 16 ) when compared to control mice ( n = 13 ; two - , unpaired t - , * p < 0 . 05 ) . Data information : Bars error bar indicates mean ± .

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D. The cumulative score of hippocampus-dependent search strategies during Barnes maze training is impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). E. Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). Data information: Bars and error bar indicates mean ± SEM.


A . Hypergeometric overlap comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in hippocampal CA1 region of Kat2A , Kmt2A Kmt2b knock out mice . Fisher ' s hypergeometric , Benjamini - Hochberg ( BH ) correction .

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A. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in the hippocampal CA1 region of Kat2A, Kmt2A and Kmt2b knock out mice. Fisher's hypergeometric test, Benjamini-Hochberg (BH) correction.


E . Cumulative hippocampus - dependent strategy scores during Barnes maze training ( One - way ANOVA ) . n = 10 per group . F . Number of visits to escape hole during probe ( n = 10 / group , One - Way ANOVA ) . Data information : Bars error bars indicate mean ± .

E. Cumulative hippocampus-dependent strategy scores during the Barnes maze training (One-way ANOVA). n = 10 per group. F. Number of visits to the escape hole during probe test (n = 10/group, One-Way ANOVA). Data information: Bars and error bars indicate mean ± SEM.


E . H3K4me3 at TSS region of genes from module1 . Inset shows statistical comparisons among groups ( One - way ANOVA , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) . Bars error bars indicate mean ± . Number of replicates analyzed for H3K4me3 profile at TSS ( Wildtype vehicle : 3 ; CamkIIδc TG - vehicle n = 4 ; CamkIIδc TG - Vorinostat n = 4 ) .

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E. H3K4me3 at TSS region of genes from RNA module1. Inset shows statistical comparisons among groups (One-way ANOVA, ***P<0.001, ****P<0.0001). Bars and error bars indicate mean ± SEM. Number of replicates analyzed for H3K4me3 profile at TSS (Wildtype vehicle: 3; CamkIIδc TG-vehicle n = 4; CamkIIδc TG-Vorinostat n = 4).


dHepaRG - TR - A3A were infected with HBV at an MOI of 300 virions / . were treated with tetracycline ( Tet ( A3A ) ) or / 300 U / ml IFNα from day 9 to 18 p . i . cccDNA amplicons were analyzed by 3D ‑

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dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell. Cells were treated with tetracycline (Tet (A3A)) or/and 300 U/ml IFNα from day 9 to 18 p.i. cccDNA amplicons were analyzed by 3D‑PCR


dHepaRG were infected with HBV at an MOI of 100 virions / treated 9 days later with 300 U / ml IFNα ( B ) or 200 U / ml IFNγ ( C ) for indicated times ( h : hours ) . Expression of indicated genes was analyzed by qRT - PCR relative to TATA - box binding ( TBP ) mRNA ( n = 6 biological replicates of two independent experiments ) .

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dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated 9 days later with 300 U/ml IFNα (B) or 200 U/ml IFNγ (C) for indicated times (h: hours). Expression of indicated genes was analyzed by qRT-PCR relative to TATA-box binding protein (TBP) mRNA (n=6 biological replicates of two independent experiments).


Liver tissue samples obtained from HBV - negative undergoing metastasis resection ( Control ) , or with acute , self - limiting HBV infection or chronic hepatitis B , or chronic coinfection with HBV hepatitis D ( HDV ) were stained for ISG20 by immunohistochemistry . For each clinical entity , tissue sections from three different are shown ; scale bar : 50 µm . ISG20 positive of each sample ( n = 3 biological replicates ) was determined by Tissue IA image software is given in % of total tissue scanned .

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Liver tissue samples obtained from HBV-negative patients undergoing metastasis resection (Control), or patients with acute, self-limiting HBV infection or chronic hepatitis B, or chronic coinfection with HBV and hepatitis D virus (HDV) were stained for ISG20 by immunohistochemistry. For each clinical entity, tissue sections from three different patients are shown; scale bar: 50 µm. ISG20 positive area of each sample (n=3 biological replicates) was determined by Tissue IA image analysis software and is given in % of total tissue area scanned.


dHepaRG were infected with HBV at an MOI of 100 virions / treated with 600 U / ml IFNα or 400 U / ml IFNγ from day 10 to 14 p . i . ISG20 was stained by immunofluorescence . human hepatocytes were infected with HBV at an MOI of 100 virions / treated with 500 U / ml IFNα or 200 U / ml IFNγ from day 4 to 6 p . i . ISG20 was stained by immunofluorescence .

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dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 10 to 14 p.i. and ISG20 was stained by immunofluorescence. Primary human hepatocytes were infected with HBV at an MOI of 100 virions/cell and treated with 500 U/ml IFNα or 200 U/ml IFNγ from day 4 to 6 p.i. and ISG20 was stained by immunofluorescence.


dHepaRG were infected with HBV at an MOI of 500 virions / , treated with 600 U / ml IFNα or 400 U / ml IFNγ from day 7 to 9 p . i . stained by immunofluorescence for ISG20 PML ( C ) or ISG20 nucleophosmin ( D ) .

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dHepaRG cells were infected with HBV at an MOI of 500 virions/cell, treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 7 to 9 p.i. and stained by immunofluorescence for ISG20 and PML (C) or ISG20 and nucleophosmin (D).


dHepaRG were infected at an MOI of 100 virions / transduced at day 7 p . i . with an adenoviral vector for expression of shRNA targeting ISG20 ( AdV - shISG20 ) . After 2 days , were treated with 300 U / ml IFNα or 200 U / ml IFNγ . ISG20 expression was analyzed by western blot after 7 days of IFNα or 12 days of IFNγ treatment , respectively .

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dHepaRG cells were infected at an MOI of 100 virions/cell and transduced at day 7 p.i. with an adenoviral vector for expression of shRNA targeting ISG20 (AdV-shISG20). After 2 days, cells were treated with 300 U/ml IFNα or 200 U/ml IFNγ. ISG20 expression was analyzed by western blot after 7 days of IFNα or 12 days of IFNγ treatment, respectively.


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

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

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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 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 ) .

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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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 .

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(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 :

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(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 .

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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 ) .

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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 .

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(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 .

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(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 .

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(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 ) .

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(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 )

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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 )

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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 )

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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 )

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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 )

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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 ) ] .

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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

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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

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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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)